Gene expression profiling identifies responsive patients with cancer of unknown primary treated with carboplatin, paclitaxel, and everolimus: NCCTG N0871 (alliance).

BACKGROUND: Carboplatin (C) and paclitaxel (P) are standard treatments for carcinoma of unknown primary (CUP). Everolimus, an mTOR inhibitor, exhibits activity in diverse cancer types. We did a phase II trial combining everolimus with CP for CUP. We also evaluated whether a gene expression profiling (GEP) test that predicts tissue of origin (TOO) could identify responsive patients. PATIENTS AND METHODS: A tumor biopsy was required for central confirmation of CUP and GEP. Patients with metastatic, untreated CUP received everolimus (30 mg weekly) with P (200 mg/m(2)) and C (area under the curve 6) every 3 weeks. The primary end point was response rate (RR), with 22% needed for success. The GEP test categorized patients into two groups: those having a TOO where CP is versus is not considered standard therapy. RESULTS: Of 45 assessable patients, the RR was 36% (95% confidence interval 22% to 51%), which met criteria for success. Grade ≥3 toxicities were predominantly hematologic (80%). Adequate tissue for GEP was available in 38 patients and predicted 10 different TOOs. Patients with a TOO where platinum/taxane is a standard (n = 19) tended to have higher RR (53% versus 26%) and significantly longer PFS (6.4 versus 3.5 months) and OS (17.8 versus 8.3 months, P = 0.005), compared with patients (n = 19) with a TOO where platinum/taxane is not standard. CONCLUSIONS: Everolimus combined with CP demonstrated promising antitumor activity and an acceptable side-effect profile. A tumor biomarker identifying TOO may be useful to select CUP patients for specific antitumor regimens. CLINICALTRIALSGOV: NCT00936702.

Design and fabrication of a hybrid alginate hydrogel/poly(ε-caprolactone) mold for auricular cartilage reconstruction.

The aim of this study was to design and manufacture an easily assembled cartilage implant model for auricular reconstruction. First, the printing accuracy and mechanical properties of 3D-printed poly-ε-caprolactone (PCL) scaffolds with varying porosities were determined to assess overall material properties. Next, the applicability of alginate as cell carrier for the cartilage implant model was determined. Using the optimal outcomes of both experiments (in terms of (bio)mechanical properties, cell survival, neocartilage formation, and printing accuracy), a hybrid auricular implant model was developed. PCL scaffolds with 600 µm distances between strands exhibited the best mechanical properties and most optimal printing quality for further exploration. In alginate, chondrocytes displayed high cell survival (~83% after 21 days) and produced cartilage-like matrix in vitro. Alginate beads cultured in proliferation medium exhibited slightly higher compressive moduli (6 kPa) compared to beads cultured in chondrogenic medium (3.5 kPa, p > .05). The final auricular mold could be printed with 300 µm pores and high fidelity, and the injected chondrocytes survived the culture period of 21 days. The presented hybrid auricular mold appears to be an adequate model for cartilage tissue engineering and may provide a novel approach to auricular cartilage regeneration for facial reconstruction. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1711-1721, 2019.

Antiintegrin alpha v beta 3 blocks human breast cancer growth and angiogenesis in human skin.

Angiogenesis plays a fundamental role in human breast tumor progression. In fact, recent findings indicate that vascular density is a prognostic indicator of breast cancer disease status. Evidence is presented that the integrin alpha v beta 3 is not only a marker of human breast tumor-associated blood vessels, but that it plays a significant role in human angiogenesis and breast tumor growth. To assess the role of alpha v beta 3-dependent angiogenesis in the progression of human breast cancer, we examined a SCID mouse/human chimeric model with transplanted full thickness human skin containing alpha v beta 3-negative human breast tumor cells. This tumor induced a human angiogenic response as measured by vascular cell immunoreactivity with monoclonal antibodies LM609 and P2B1 directed to human alpha v beta 3 and CD31, respectively. Intravenous administration of LM609 either prevented tumor growth or markedly reduced tumor cell proliferation within the microenvironment of the human skin. These LM609-treated tumors not only contained significantly fewer human blood vessels but also appeared considerably less invasive than tumors in control animals. These findings demonstrate that alpha v beta 3 antagonists may provide an effective antiangiogenic approach for the treatment of human breast cancer.

A case report on a burned ear: Elastic memory of cartilage following temporary burial in a skin pocket.

Preserving exposed ear cartilage following a facial burn remains a major challenge. Normally, burned ear cartilage cannot be preserved in case of a full thickness burn of the overlying skin, and the cartilage has to be surgically removed. Sometimes, reconstructions can be performed at a later stage. We report a case where burned ear cartilage was directly surgically buried in a retroauricular skin pocket showing remarkable elastic memory: the buried ear cartilage, in this case the antihelix, regenerated over time and regained its original position protruding from the facial area. This case illustrates that ear cartilage is highly resilient, even when it has sustained significant thermal damage, and can be buried in a retroauricular skin pocket to avoid radical excision of the framework.

Maspin expression inversely correlates with breast tumor progression in MMTV/TGF-alpha transgenic mouse model.

Maspin is a novel serine protease inhibitor (serpin) with tumor suppressive activity. To date, despite the mounting evidence implicating the potential diagnostic/prognostic and therapeutic value of maspin in breast and prostate carcinoma, the lack of a suitable animal model hampers the in vivo investigation on the role of maspin at different stages of tumor progression. In this study, we used MMTV/TGF-alpha transgenic mouse model to study the expression profile of maspin in mammary tumor progression. Histopathological examinations of MMTV/TGF-alpha transgenic mice revealed TGF-alpha expression leading to hyperproliferation, hyperplasia, and occasional carcinoma in mammary gland. Interestingly, when MMTV/TGF-alpha transgenic mice were breed to homozygocity, they also developed characteristic skin papillomas. Immunohistochemistry analysis of maspin expression in the breast tissues of TGF-alpha transgenic mice showed a direct correlation between down-regulation of maspin expression and tumor progression. The loss of maspin expression was concomitant with the critical transition from carcinoma in situ to invasive carcinoma. Subsequent in-situ hybridization analyses suggest that the down-regulation of maspin expression is primarily a transcriptional event. This data is consistent with the tumor suppressive role of maspin. Furthermore, our data suggests that MMTV/TGF-alpha transgenic mouse model is advantageous for in vivo evaluation of both the expression and the biological function of maspin during the slow multi-stage carcinogenesis of mammary gland.

Inhibition of murine renal carcinoma pulmonary metastases by systemic administration of interferon gamma: mechanism of action and potential for combination with interleukin 4.

We have previously demonstrated that IFN-gamma causes cell growth inhibition and up-regulation of MHC antigens in human renal cell carcinoma cell lines. In this study, we have investigated the therapeutic potential of IFN-gamma for the treatment of 5-day established pulmonary metastases induced by i.v. injection of Renca cells, a murine renal adenocarcinoma. We found that systemic injections of IFN-gamma significantly reduced the number of lung metastases in a dose-dependent manner and increased mouse survival. Histological evaluation of IFN-gamma-treated lungs showed residual small tumor nodules containing extensive necrosis and mononuclear infiltrates. Immunohistochemistry studies on lung sections showed macrophage infiltration into tumor nodules, and in vivo depletion of macrophages partially inhibited IFN-gamma antitumor effect, suggesting a role for the macrophages in tumor destruction. Lymphocyte depletion of either natural killer (NK) cells or CD4+ or CD8+ T-cell subsets or both T-cell subsets did not affect the IFN-gamma effect, whereas depletion of both NK and T cells decreased the antitumor activity of IFN-gamma. These data indicate that neither T cells nor NK cells are essential for this activity but that either lymphocyte population can contribute to the IFN-gamma effect. An optimal dose of IFN-gamma inhibited by 60% the growth of Renca cells treated for 3 days in vitro, but this effect was transient and less pronounced in a long-term colony assay, suggesting that IFN-gamma direct growth inhibition may play a role but may not be sufficient to mediate its antitumor effect in vivo. In vitro, IFN-gamma caused up-regulation of class I MHC antigens and induction of class II antigen expression in Renca cells, an effect that may enhance Renca immunogenicity but may be relevant only when a T-cell response is elicited. A sequential administration of IFN-gamma followed by interleukin 4 was therapeutically better than IFN-gamma alone for the treatment of advanced pulmonary metastases, probably due to different antitumor mechanisms induced by these two cytokines.

Centrally necrotizing carcinomas of the breast: a distinct histologic subtype with aggressive clinical behavior.

Most breast carcinomas exhibit ductal differentiation. However, recognition of less common histologic patterns provides clinically useful data. This report describes a distinctive subtype of breast carcinoma that we have termed "centrally necrotizing carcinoma" (CNC; in this study, N = 34), which is characterized by an unusual and aggressive natural history. Centrally necrotizing carcinomas are composed of well-circumscribed, unicentric nodules with extensive central necrosis that are surrounded by a narrow rim of viable high-grade tumor cells. These tumor cells show minimal ductal differentiation (i.e., tubule formation), but are usually associated with focal ductal carcinoma in situ. The mean age of the patients in this study was 57.5 +/- 11.6 years, and the mean tumor size was 2.5 +/- 1.2 cm. Twenty-eight percent of the patients had positive axillary lymph nodes (mean number of lymph nodes involved, 2.1 +/- 1.2). Ninety-four percent of cases were negative for estrogen and progesterone receptors. In 21 patients (62%), local and/or distant recurrences developed (median time to recurrence, 16.2 months), and, to date, 20 have died from breast cancer (median time to death, 22.5 months). Progression of disease (defined as the development of either a recurrence or death resulting from disease) occurred in 24 patients (71%). Comparison with a set of 26 poorly differentiated ductal carcinomas with (nonextensive, patchy) necrosis matched for age, tumor size, and lymph node status showed a significantly worse progression-free survival rate for the CNC group (p < 0.004). We conclude that CNC is an uncommon but readily identifiable subtype of breast carcinoma and is characterized by early systemic metastasis and an accelerated clinical course.

Clinicopathologic significance of ductal carcinoma in situ in breast core needle biopsies with invasive cancer.

To assess whether the presence and amount of intraductal component (IC) in diagnostic needle core biopsies (NCB) is predictive of an extensive IC (EIC), the authors evaluated 50 invasive ductal carcinomas diagnosed with NCB, and then excised via lumpectomy, with regard to the extent of IC in both the NCB and subsequent lumpectomy specimen. These parameters were compared with each other and with the lumpectomy margin status. Extent of IC in the NCB was evaluated by dividing the number of ducts that contained IC by the total number of tissue cores. A ratio of more than 0.5 was considered EIC (EICc). IC extent in the lumpectomy was established by estimating the percentage of the tumor corresponding to IC and was considered extensive (EIC(L)) if more than 25% and if there was presence of IC away from the invasive tumor. The mean size of resected tumors was 1.6 +/- 0.7 cm. In 29 cases (58%) there was no IC in the NCB (NegICc), 11 cases (22%) exhibited nonextensive IC (NEICc), and 10 cases (20%) demonstrated EICc. A total of 7%, 36%, and 70% of the NegICc, NEICc, and EICc cases respectively had EIC(L)(p < 0.0001). The presence of EIC(L) correlated significantly with close or positive margin status for in situ disease (EIC(L) positive, 12 of 13 [92%] vs EIC(L) negative, 11 of 37 [30%]; p = 0.004). None of the NegICc, 27% of NEICc, and 40% of EICc had a positive margin for in situ neoplasm in the lumpectomy specimen (p = 0.004), and 24%, 18%, and 50% had positive margins for invasive neoplasm (p = not significant). The authors conclude that EICc predicts EIC(L) and constitutes a risk factor for positive lumpectomy margin status-particularly for in situ tumor. EICc may thus be of clinical value in identifying a subset of patients that requires a wider local excision.

Clear cell tumor of the lung. A clinicopathologic, immunohistochemical, and ultrastructural study of eight cases.

We studied eight clear cell tumors of the lung (CCTL) to better define their clinical, immunohistochemical, and ultrastructural features, and to clarify their distinction from other neoplasms, particularly metastatic renal cell carcinoma. Patients ranged in age from 31 to 67 years (mean, 51 years). Seven patients had clinically benign, asymptomatic lesions measuring less than 2 cm in diameter that were devoid of necrosis. The eighth patient had a symptomatic, partially necrotic CCTL 4.5 cm in diameter that metastasized to the liver and peritoneum; the patient died of tumor 17 years after diagnosis. Ultrastructural study of seven CCTL showed interdigitating cell processes (all cases), primitive cell junctions (five of seven cases), intracytoplasmic glycogen (all cases), and rare dense core granules (two of seven cases). Immunohistochemically, paraffin-embedded sections from all eight CCTL were negative for cytokeratin (CK), epithelial membrane antigen (EMA), chromogranin, and vimentin. Focal staining was seen for S-100 protein (three of eight cases), neuron-specific enolase (three cases), synaptophysin (one case), and Leu 7 (one case). Although these findings suggest that at least some CCTL exhibit neuroendocrine differentiation, the tumor's histogenesis remains uncertain. Of more practical importance, the combined absence of CK, EMA, and vimentin in formalin-fixed, paraffin-embedded CCTL virtually precludes confusion with renal cell carcinoma. Although traditionally considered benign, CCTL larger than 2 cm that are symptomatic, and focally necrotic should be regarded as potentially malignant neoplasms.

Histologic and immunophenotypic evaluation of pretreatment renal biopsies in OKT3-treated allograft rejections.

The histologic features immunophenotype of intragraft mononuclear populations, and HLA-Dr expression in pretreatment formalin-fixed renal allograft biopsies were correlated with outcome of OKT3 therapy in 35 steroid resistant renal transplant rejections. Therapeutic response (63% overall) was better in pure acute cellular rejections (ACR) (13/15, 87%) than ACR with interstitial fibrosis (4/12, 33%) or with vascular injury (5/8, 62%). Intragraft T lymphocytes were more numerous in vascular rejection (mean 566/mm2) compared with pure ACR (mean 265/mm2, P = .049), and macrophages were greater in pure ACR (203/mm2) compared with ACR with interstitial fibrosis (83/mm2, P = .051). Distribution of T cells, B cells, plasma cells, and macrophages among various histologic categories was otherwise statistically similar. There was no correlation between therapeutic response to OKT3 and intragraft concentrations of individual mononuclear cell subsets. Vascular and/or epithelial HLA-Dr expression was present in 17/25 (68%) cases and was not associated with histologic features or treatment response. Follow-up graft function (median 7 months) correlated significantly with therapeutic response to OKT3 (P = .0004) and histologic presence of interstitial fibrosis (P = .031), but was not related to concentration of individual mononuclear subsets or HLA-Dr expression. We conclude that intragraft concentrations of major mononuclear cell types may relate to histology, but that these do not predict treatment response or graft outcome, and thus poorly reflect intensity or possible heterogeneity of host immunologic rejection mechanisms.

Clinicopathologic analysis of microscopically invasive breast carcinoma.

Breast biopsy or mastectomy cases having diagnoses of carcinoma in situ with "microinvasion," "minimal invasion," "focal invasion," or "suggestive of invasion" were reviewed and all histologically identified foci of invasive disease from each case were measured using an ocular micrometer. Cases in which any single focus of invasion was greater than 5 mm or the added size of separate invasive foci exceeded 10 mm were excluded, resulting in a study group of 75 patients. Invasive neoplasm was present in the initial biopsy in 69 of 75 cases (92%); however, residual invasive neoplasm was found in the subsequent lumpectomy/mastectomy from 14 of these (20%). In 59% of cases, two or more histologically separate foci of invasion were identified. Invasive foci consisted of isolated cells or cell clusters, each less than 1 mm (microfocal invasion), in 33% of cases. In 12 cases, the sum of individual invasive foci was 5 to 10 mm. Axillary lymph nodes (LN) from 5 of 69 patients (7%) contained metastatic carcinoma (four cases, one LN positive; one case, two LN positive). The cumulative sizes of all invasive foci in the LN-positive group were microfocal invasion (one case), 0.6 mm (one case), 1.1 mm, 2.5 mm, and 5.8 mm. The difference in frequency of axillary node metastasis between tumors with microfocal and measurable invasion (4.3% v 8.6%) was not statistically significant. Follow-up data were available on 55 cases (mean interval, 66.1 months). One (node-negative) patient had duct carcinoma in situ recurrence in the same breast 4 years after initial treatment. Another (with unknown node status) developed an axillary lymph node metastasis 13 months after initial treatment (96% disease-free survival). We conclude that microscopic stromal invasion in breast carcinoma, at least in the setting of significant in situ component, is often initiated from multiple foci. Patients with microscopically invasive breast carcinoma have a small but significant risk of axillary metastases, although a highly favorable survival.

Histopathologic analysis of chromosome aneuploidy in ductal carcinoma in situ.

Formalin-fixed, paraffin-embedded sections from 28 cases of ductal carcinoma in situ (DCIS; 12 with coexisting invasive neoplasm) were analyzed for numerical alterations of chromosomes 7, 8, 16, and 17 by performing fluorescence in situ hybridization (FISH) using centromeric (alpha-satellite) probes. Based on signal counts in 200 to 300 nuclei, each hybridization was classified as disomic (copy loss in <40%, copy gain in < 10%), monosomic (copy loss in at least 50% of nuclei, partial if 40% to 49%) or trisomic/polysomic (copy gain in at least 20% of nuclei, partial if 10% to 19%). Grade I lesions were characterized by complete lack of significant chromosome gain, but 29% showed partial (focal) monosomy. Grade III lesions, in contrast, showed partial or complete trisomy/polysomy in 88% of hybridizations versus monosomy in only 4%. Grade II DCIS exhibited a mixed pattern of chromosome aneuploidy: 38% hybridizations were disomic, 36% trisomic/polysomic, and 26% monosomic (8 of 10 hybridizations showing complete monosomy occurred in grade II lesions). Disomic hybridizations exhibiting rare cells (5% to 10%) with copy gain were more frequent in tumors with coexisting invasive neoplasm (5 of 17 v 2 of 33, P = .02). In morphologically heterogeneous lesions, higher-grade foci were characterized by chromosome copy gain relative to corresponding lower-grade areas in 17 of 22 (77%) hybridizations. These results show the presence of multiple (at least 3) distinct chromosome aneuploidy patterns in DCIS, in keeping with divergent mechanisms of genetic alteration. Degree of chromosomal instability, moreover, may correlate with progression of DCIS to invasive growth, implying that genetic instability is a parameter that impacts the likelihood of early breast carcinoma progression.

Chromosomal aneuploidy in proliferative breast disease.

Although some forms of proliferative breast disease have been associated with increased risk of breast cancer, substantial confirmatory evidence that the lesions are biologically premalignant has not been presented. Our intent was to identify cytogenetic aberrations in proliferative breast disease using fluorescence in situ hybridization probes selected for their relationship to aberrations previously reported in breast cancer. Application of fluorescence in situ hybridization techniques to paraffin tissue sections using pericentromeric probes for chromosomes 1, 16, 17, 18, and X revealed chromosome aneuploidy in proliferative and malignant lesions of the breast. Sectioning artifact that may result in nuclear truncation was controlled by establishing expected baseline frequencies for gain and loss in normal tissues from the same breast. Localization of chromosomal aberrations to proliferative breast disease lesions with concomitant retention of a normal chromosome complement in corresponding normal breast tissues indicates biologic significance of the results. The similarities of losses involving chromosomes 16, 17, and 18 in hyperplastic lesions and in malignant breast lesions suggest that some hyperplasias may be part of a sequence of progression to malignancy in breast cancer. Gains of chromosome 1 in both in situ and invasive carcinoma are consistent with reports of polysomy 1q as a common cytogenetic change in breast cancer. Its localization to advanced lesions suggests that this trisomy is probably not the initial cytogenetic change in breast cancer tumorigenesis.

Idiopathic bronchiolitis obliterans with organizing pneumonia. An acute and life-threatening syndrome.

Idiopathic bronchiolitis obliterans with organizing pneumonia (BOOP) is a clinicopathologic syndrome characterized by an indolent course and favorable prognosis. This report describes five patients with a fulminating and life-threatening variant of this syndrome. Four patients presented with respiratory failure requiring respiratory assistance and positive pressure ventilation. Early recognition of the entity and prompt initiation of corticosteroid therapy in three patients was instrumental in preventing mortality. Our findings suggest that idiopathic BOOP may be the underlying pathology in a number of patients presenting with ARDS. Since corticosteroid therapy may improve survival in these patients, clinicians should heighten their index of suspicion for this entity. Early histologic diagnosis and initiation of corticosteroid therapy should be considered in patients with unexplained ARDS.

Coordinate expression of the PRM1, PRM2, and TNP2 multigene locus in human testis.

Maintenance of the transcriptionally inert state of the mature human spermatozoon requires the expression of the various members of the human protamine gene cluster prior to the final stages of spermatogenesis. During this process, known as spermiogenesis, round spermatids morphologically differentiate into mature spermatozoa. The expression of the PRM1, PRM2, and TNP2 genes facilitates the compaction and condensation of the genetic material within the developing spermatid. To understand better the coordinate control governing this transformation, we have examined the localization and distribution of the human protamines PRM1 and PRM2 and transition protein TNP2 transcripts during human spermatogenesis. The stage-specific expression of these transcripts was determined by in situ hybridization analysis using [alpha-35S]-labeled cRNA probes. PRM1, PRM2, and TNP2 transcripts were abundant in association with round and elongating spermatids, located in the adluminal region of the seminiferous epithelium. They were not observed in association with spermatogonia, spermatocytes, Sertoli cells, or interstitial cells. These data indicate that the human PRM1, PRM2, and TNP2 transcripts are expressed postmeiotically in round and elongating spermatids. The quantitative evaluation of each transcript was determined as a function of the relative optical density per unit area. In all cases examined, the relative level of each transcript was consistent with the following pattern, PRM2 > PRM1 congruent to TNP2.

Quantitative analysis of Her-2/neu (ERBB2) gene expression using reverse transcriptase polymerase chain reaction.

Inappropriate expression of Her-2/neu (ERBB2) gene has been associated with impaired breast cancer prognosis, suggesting a functional role in tumor progression. Herein we describe a quantitative method for analysis of Her-2/neu gene messenger RNA (mRNA), which employs reverse transcriptase polymerase chain reaction (RT-PCR) on a 10-microns cryostat section. The technique combines modified RNA extraction with complementary DNA (cDNA) synthesis to achieve a high level of sensitivity. Utilizing this PCR-based gene expression assay, we were able to quantitate variable amounts of Her-2/neu mRNA in cell lines with established levels of gene expression and in clinical human breast cancer specimens. In clinical samples, mRNA levels correlated with intensity of immunoperoxidase staining for corresponding oncoprotein. We conclude that PCR-based mRNA quantitation can be applied to quantitative analysis of Her-2/neu gene expression, and potentially many other genes, in samples of limited size.

Clinicopathologic analysis of k-ras, p53, and ERBB-2 gene alterations in pulmonary adenocarcinoma.

We compared PCR-SSCP detected mutations of k-ras (codon 12) and p53 (exons 5-8) to ERBB-2 immunostaining and clinicopathologic features in 31 pulmonary adenocarcinomas. There were nine tumors (29%) with mutations of ras, 13 tumors (42%) with mutations of p53, and three tumors (10%) with mutations of both. Neither k-ras nor p53 mutation alone was significantly correlated with stage, grade, or survival. However, tumors with k-ras mutation were more frequently associated with an invasive growth pattern, defined as > 30% tumor volume composed of infiltrative nests of cells within desmoplastic, scar-like stroma [< 30% volume invasive--1/13 (8%) with k-ras mutation vs. > 30% volume invasive--8/18 (44%) with k-ras mutation, p = 0.02]. Accordingly, k-ras mutations were observed in only 1/9 (15%) predominantly bronchoalveolar or papillary tumors versus 6/22 (28%) acinar or scar carcinoma tumors. All three patients with combined k-ras/p53 mutation had advanced stage (III/IV) at presentation and died of the disease. In contrast to k-ras, staining for ERBB-2 was more frequently observed in tumors exhibiting < 30% invasive growth pattern (12/13, 92%) than in tumors with > 30% invasive growth pattern (10/18, 56%, p = 0.03). ERBB-2 immunoreactivity was more frequent in Stage I (14/15, 93%) versus Stage II-IV (8/16, 50%) cases, but it did not correlate with survival. There was a reciprocal relationship between k-ras mutation and ERBB-2 staining; only 4/9 (44%) k-ras mutated cases were ERBB-2 positive versus 18/22 (82%) cases without k-ras mutation (p = 0.005). In contrast, 8/13 cases with p53 mutation were ERBB-2 positive. We conclude that well-differentiated and less invasive papillary and bronchoalveolar tumors are more often ERBB-2 positive/k-ras negative (i.e. at codon 12), whereas less well differentiated acinar or scar carcinomas are more often ERBB-2 negative/k-ras mutated at codon 12. These findings imply that the divergent histogenesis of pulmonary adenocarcinoma may reflect specific differences in genetic pathology.

Correlation between p53 immunostaining patterns and gene sequence mutations in breast carcinoma.

We performed p53 immunostaining in 82 invasive breast carcinomas by using two commercially available antibodies, one of which (DO7) was employed in formalin-fixed paraffin-embedded sections. The other antibody (PAb1801) was evaluated in corresponding acetone-fixed cryostat sections. A greater percent of cases were immunostained with DO7 compared to PAb1801 (52% vs 33%); however, the staining was more often heterogeneous (6-50% cells positive) or focal (< or = 5% cells positive) with DO7 (9% vs 31%). To investigate the genetic relevance of p53 immunostaining, single-strand conformational polymorphism (SSCP) analysis and DNA sequencing were performed on exons 2-11 by using archival tissue samples of 18 cases that were selected on the basis of certain immunostaining patterns. Two (33%) of six tumors with negative staining for DO7 had gene sequence mutations; however, one of these mutations was a base-pair deletion that caused a reading-frame shift and the other was a base-pair insertion that resulted in a stop codon. Both of these tumors exhibited immunostaining with PAb1801, although it was weak and cytoplasmic in one case. Conversely, three (30%) of 10 tumors showing immunoreactivity in 6-100% of cells with both reagents lacked a gene sequence mutation. Of the remaining seven tumors that were positive by SSCP, six contained a point mutation resulting in a base-pair substitution. Despite repeat analyses, one of the cases positive by SSCP failed to demonstrate a mutation in the sequenced exons. Four (80%) of five cases with heterogeneous DO7 immunoreactivity (that is, 6-50% of nuclei positive) were positive for gene sequence mutation. Neither of two cases showing focal DO7 nuclear staining in < 5% of tumor cells contained a mutation in the sequenced exons, and neither of these cases was strongly positive with PAb1801. Staining for either antibody was significantly associated with adverse outcome, as determined by disease recurrence at 52 months median follow-up (DO7, p = 0.01; and PAb1801 p = 0.002, chi-squared test). We conclude that a variety of factors may account for discrepancies when immunohistology is used to evaluate p53 status. These include fixation artifacts, differing epitope specificities of monoclonal reagents, presence of immunohistologically "silent" mutations and, possibly, aberrant overexpression of wild-type protein.

A universal method for the mutational analysis of K-ras and p53 gene in non-small-cell lung cancer using formalin-fixed paraffin-embedded tissue.

The p53 tumor suppressor gene has been found to be altered in almost all human solid tumors, whereas K-ras gene mutations have been observed in a limited number of human cancers (adenocarcinoma of colon, pancreas, and lung). Studies of mutational inactivation for both genes in the same patient's sample on non-small-cell lung cancer have been limited. In an effort to perform such an analysis, we developed and compared methods (for the mutational detection of p53 and K-ras gene) that represent a modified and universal protocol, in terms of DNA extraction, polymerase chain reaction (PCR) amplification, and nonradioisotopic PCR-single-strand conformation polymorphism (PCR-SSCP) analysis, which is readily applicable to either formalin-fixed, paraffin-embedded tissues or frozen tumor specimens. We applied this method to the evaluation of p53 (exons 5-8) and K-ras (codon 12 and 13) gene mutations in 55 cases of non-small-cell lung cancer. The mutational status in the p53 gene was evaluated by radioisotopic PCR-SSCP and compared with PCR-SSCP utilizing our standardized nonradioisotopic detection system using a single 6-microns tissue section. The mutational patterns observed by PCR-SSCP were subsequently confirmed by PCR-DNA sequencing. The mutational status in the K-ras gene was similarly evaluated by PCR-SSCP, and the specific mutation was confirmed by Southern slot-blot hybridization using 32P-labeled sequence-specific oligonucleotide probes for codons 12 and 13. Mutational changes in K-ras (codon 12) were found in 10 of 55 (18%) of non-small-cell lung cancers. Whereas adenocarcinoma showed K-ras mutation in 33% of the cases at codon 12, only one mutation was found at codon 13. As expected, squamous cell carcinoma samples (25 cases) did not show K-ras mutations. Mutations at exons 5-8 of the p53 gene were documented in 19 of 55 (34.5%) cases. Ten of the 19 mutations were single nucleotide point mutations, leading to amino acid substitution. Six showed insertional mutation, and three showed deletion mutations. Only three samples showed mutations of both K-ras and p53 genes. We conclude that although K-ras and p53 gene mutations are frequent in non-small-cell lung cancer, mutations of both genes in the same patient's samples are not common. We also conclude that this universal nonradioisotopic method is superior to other similar methods and is readily applicable to the rapid screening of large numbers of formalin-fixed, paraffin-embedded or frozen samples for the mutational analysis of multiple genes.

Influence of tumor site on the therapy of murine kidney cancer.

OBJECTIVE: The purpose of this study was to evaluate the influence of tumor site on the therapy of an experimental murine kidney cancer model, Renca. METHODS: Equal number of tumor cells were injected subcutaneously, intraperitoneally, sub-renal capsule, and intravenously to induce tumors. The animals were then assessed for growth of the primary tumor, metastases and survival. Immunohistochemistry and flow cytometry was performed to identify the phenotype of infiltrating lymphocytes. Tumor bearing animals were treated with high dose interleukin-2 or whole body radiation, and response of the primary tumors as well as the metastases was assessed. RESULTS: Renca tumors grew well regardless of the methods of induction. Spontaneous metastasis could be best induced in the sub-renal capsule model. More consistent numbers of pulmonary metastases were obtained by intravenous injection of the tumor cells. Immunotherapy was able to reduce the size of the primary tumor as well as the number of metastasis. Whole body radiation caused some reduction in the primary tumor but did not have a major effect in reducing metastasis. CONCLUSIONS: The Renca model is a suitable animal model to study the response to different therapeutic interventions. The site of the tumor growth is not a significant variable in the response to treatment.