Desmond N. Carney

The International Association for the Study of Lung Cancer Lung Cancer Staging Project: Proposals Regarding the Clinical Staging of Small Cell Lung Cancer in the Forthcoming (Seventh) Edition of the Tumor, Node, Metastasis Classification for Lung Cancer

Background: Small cell lung cancer (SCLC) is usually classified using the limited and extensive definition. The tumor, node, metastasis (TNM) classification should also be applicable to SCLC, but it has only been reported in small surgical series. The current analysis looks to the impact of the TNM system on the clinical staging of SCLC and of the new International Association for the study of Lung Cancer (IASLC) proposals. Methods: Using the IASLC database, survival analyses were performed for clinically staged patients. Prognostic groups were compared, and the new IASLC TNM proposals were applied to this population and to the Surveillance, Epidemiology, and End Results (SEER) database. Results: The IASLC database contained 12,620 eligible cases of small cell histology. TNM staging was available for 8088 patients. Survival was directly correlated to both T and N category. Differences were more pronounced in patients without mediastinal or supraclavicular nodal involvement. Stage grouping using the sixth edition of TNM also differentiates survival except between IA and IB. Patients with pleural effusion regardless of the cytology have an intermediate prognosis between limited and extensive disease. The IASLC proposals for the seventh edition of the TNM classification also apply to this series of SCLC and to the SEER database. Conclusion: TNM staging is recommended for SCLC, and stratification by stage I-III should be incorporated in clinical trials of early-stage disease. Further studies are needed to clarify the impact of pleural effusion and the extent of N3 disease.

The IASLC Lung Cancer Staging Project: Proposals Regarding the Relevance of TNM in the Pathologic Staging of Small Cell Lung Cancer in the Forthcoming (Seventh) Edition of the TNM Classification for Lung Cancer

Introduction: For more than 50 years, small cell lung cancer (SCLC) has been staged mainly as either limited or extensive stage disease. Small published series of resected SCLC have suggested that the tumor, node, metastases (TNM) pathologic staging correlates with the survival of resected patients. Recent analysis of the 8088 cases of SCLC in the International Association for the Study of Lung Cancer (IASLC) database demonstrated the usefulness of clinical TNM staging in this malignancy. The IASLC data bank contains an unprecedented number of resected SCLC cases with pathologic staging information. This analysis was undertaken to examine the impact of the TNM system on the pathologic staging of SCLC and to assess the new IASLC proposals in this subtype of lung cancer. Methods: Using the IASLC database, survival analyses were performed for resected patients with SCLC. Prognostic groups were compared, and the new IASLC TNM proposals were applied to this population and to the Surveillance, Epidemiology, and End Results (SEER) database. Results: The IASLC database contained 349 cases of resected SCLC where pathologic TNM staging was available. Survival after resection correlated with both T and N category with nodal status having a stronger influence on survival. Stage groupings using the 6th edition of TNM clearly identify patient subgroups with different prognoses. The IASLC proposals for the 7th edition of TNM classification also apply to this population and to the SEER database. Conclusion: This analysis further strengthens our previous recommendation to use TNM staging for all SCLC cases.

A nonrandom chromosomal abnormality, del 3p(14-23), in human small cell lung cancer (SCLC).

In order to determine whether or not there are specific chromosomal changes in small cell lung cancer (SCLC), karyotypic analyses of 16 continuous SCLC tissue culture lines, three fresh tumor specimens (bone marrow), one direct preparation of bone marrow involved with SCLC, and two lymphoblastoid lines derived from SCLC patients were studied. Cell lines were derived from primary tumor, or metastases to bone marrow, subcutaneous nodules, or pleural fluid; all 16 lines had biochemical and histologic properties characteristic of SCLC. Of the 15 males and 3 females, 6 patients had no prior treatment. All of the 16 cell lines, the 3 fresh specimens, and the direct bone marrow preparation had a common deletion of the short arm of chromosome #3. Use of the shortest region of overlap analysis showed the common deletion was of the short arm in the regions p(14-23). This specific chromosomal abnormality, del 3p, was not found in five non-SCLC cell lines studied and is of major potential biological and diagnostic importance.

I: High affinity receptors for bombesin/GRP-like peptides on human small cell lung cancer

The binding of a radiolabeled bombesin analogue to human small cell lung cancer (SCLC) cell lines was investigated. (125I-Tyr4)bombesin bound with high affinity (Kd = 0.5 nM) to a single class of sites (2,000/cell) using SCLC line NCI-H446. Binding was reversible, saturable and specific. The pharmacology of binding was investigated using NCI-H466 and SCLC line NCI-H345. Bombesin and structurally related peptides, such as gastrin releasing peptide (GRP), but not other peptides, such as substance P or vasopressin, inhibited high affinity (125I-Tyr4)BN binding activity. Finally, the putative receptor, a 78,000 dalton polypeptide, was identified by purifying radiolabeled cell lysates on bombesin or GRP affinity resins and then displaying the bound polypeptides on sodium dodecylsulfate polyacrylamide gels. Because SCLC both produces bombesin/GRP-like peptides and contains high affinity receptors for these peptides, they may function as important autocrine regulatory factors for human SCLC.

Amplification of the MDM2 gene in human breast cancer and its association with MDM2 and p53 protein status.

The present study reports on the frequency of MDM2 gene amplification and MDM2 protein expression in a series of 100 breast carcinomas and its association with accumulation of the p53 protein. Of the 100 cases, frozen samples for 82 cases were available for Southern blotting. Three of the 82 (4%) demonstrated MDM2 gene amplification of up to 6-fold. Immunohistochemical analysis of the formalin-fixed, paraffin-embedded tumours demonstrated that 7/97 (7%) had nuclear expression for MDM2 in 10-50% of the tumour cells (type 2 staining) and were denoted MDM2+. Two of the MDM2-amplified samples were MDM2+ with one of the two tumours also displaying type 2 p53 nuclear staining. Finally at the protein level, MDM2+ tumours were significantly associated with tumours having low levels of p53 staining (0-10% cells positive) (P = 0.03). We conclude that MDM2 gene amplification occurs at a lower frequency in breast cancer than in non-epithelial tumours. Alterations in MDM2 and p53 may represent alternative pathways in tumorigenesis, but they are not mutually exclusive in all cases.

Immunohistochemical analysis of cell kinetic parameters in colonic adenocarcinomas, adenomas, and normal mucosa

The monoclonal antibody Ki-67 identifies a nuclear antigen that is expressed in proliferating cells in G1, G2, S, and M phases of the cell cycle. An immunoperoxidase method and this antibody were used to identify proliferating cells in sections of colorectal tissues--normal colon (n = 10), colorectal polyps (n = 20), and adenocarcinoma (n = 28). Colorectal adenomas showed a uniform distribution of positive nuclear staining throughout the sections, including the cells of the adenoma surface, while staining in the normal mucosa was confined to the middle third and lower third of the crypts. Areas of polyps with numerous Ki-67-positive epithelial cells invariably showed immature or dysplastic histology and, conversely, glands that lacked such histologic features had low Ki-67 staining frequency or were negative. In adenomas, nuclei located toward the luminal surface of glands were more likely to be Ki-67-positive than those located basally in the cells. The mean Ki-67 score (a measure of positive staining nuclei) for adenomas was 45.5 compared to a mean score of 66.3 for adenocarcinomas in the carcinomas studied (P less than .001). Ki-67 score did not correlate with histologic grade or Dukes stage. Ki-67 staining can be used to characterize the proliferative characteristics of normal colonic mucosa, adenomas, and carcinomas.

Changes in the phenotype of human small cell lung cancer cell lines after transfection and expression of the c-myc proto-oncogene.

Small cell lung cancer growing in cell culture possesses biologic properties that allow classification into two categories: classic and variant. Compared with classic small cell lung cancer cell lines, variant lines have altered large cell morphology, shorter doubling times, higher cloning efficiencies in soft agarose, and very low levels of L dopa decarboxylase production and bombesin-like immunoreactivity. C-myc is amplified and expressed in some small cell lung cancer cell lines and all c-myc amplified lines studied to date display the variant phenotype. To investigate if c-myc amplification and expression is responsible for the variant phenotype, a normal human c-myc gene was transfected into a cloned classic small cell lung cancer cell line not amplified for or expressing detectable c-myc messenger RNA (mRNA). Clones were isolated with one to six copies of c-myc stably integrated into DNA that expressed c-myc mRNA. In addition, one clone with an integrated neo gene but a deleted c-myc gene was isolated and in this case c-myc was not expressed. C-myc expression in transfected clones was associated with altered large cell morphology, a shorter doubling time, and increased cloning efficiency, but no difference in L dopa decarboxylase levels and bombesin-like immunoreactivity. We conclude increased c-myc expression observed here in transfected clones correlates with some of the phenotypic properties distinguishing c-myc amplified variants from unamplified classic small cell lung cancer lines.

DNA Content Analysis by Flow Cytometry and Cytogenetic Analysis in Mycosis Fungoides and Sézary Syndrome: DIAGNOSTIC AND PROGNOSTIC IMPLICATIONS

Flow cytometric (FCM) analysis of DNA content was performed on 82 lymph node and peripheral blood specimens from 46 patients with mycosis fungoides and the Sézary syndrome. Overall, 32 of the 46 patients (70%) had aneuploidy detected by FCM. Aneuploidy was present in 63% of the patients at the time of diagnosis before systemic therapy. In these patients, aneuploidy was frequently detected in blood and lymph node specimens scored as negative by cytology and histology, suggesting that unsuspected extracutaneous dissemination is present in many patients at the time of diagnosis. Direct comparison with Giemsa-banded cytogenetic studies showed an excellent correlation of FCM results and cytogenetic chromosome number. However, FCM frequently detected a larger fraction of aneuploid cells, and mitogen-stimulation studies suggest this is the result of preferential stimulation of normal lymphocytes by phytohemagglutinin. Thus, mitogens with a preference for malignant T cells, such as staphylococcal protein A, should be used for cytogenetic analysis of malignant T-cell disorders. At diagnosis, some histologically positive specimens contained only diploid cells by FCM and cytogenetic analysis. These patients had a more indolent clinical course than patients with aneuploidy. Aneuploidy was detected by FCM as either wide G(1) or as discrete aneuploid peaks. The presence of aneuploidy at any time in the clinical course implied a poor prognosis. Discrete hyperdiploid peaks were associated with large cell histology, early relapse, and aggressive clinical course. The development of hyperdiploidy at relapse was documented in four patients and was associated with a transition to large cell histology and a poor prognosis. Similar studies may elucidate differences in natural history and mechanism for transition in histology in other lymphomas and solid tumors.

A germline TaqI restriction fragment length polymorphism in the progesterone receptor gene in ovarian carcinoma

Clinical outcome in ovarian carcinoma is predicted by progesterone receptor status, indicating an endocrine aspect to this disease. Peripheral leucocyte genomic DNAs were obtained from 41 patients with primary ovarian carcinoma and 83 controls from Ireland, as well as from 26 primary ovarian carcinoma patients and 101 controls in Germany. Southern analysis using a human progesterone receptor (hPR) cDNA probe identified a germline TaqI restriction fragment length polymorphism (RFLP) defined by two alleles: T1, represented by a 2.7 kb fragment; and T2, represented by a 1.9 kb fragment and characterised by an additional TaqI restriction site with respect to T1. An over-representation of T2 in ovarian cancer patients compared with controls in the pooled Irish/German population (P < 0.025) was observed. A difference (P < 0.02) in the distribution of the RFLP genotypes between Irish and German control populations was also observed. The allele distributions could not be shown to differ significantly from Hardy-Weinberg distribution in any subgroup. Using hPR cDNA region-specific probes, the extra TaqI restriction site was mapped to intron G of the hPR gene.

Late intensive combined modality therapy followed by autologous bone marrow infusion in extensive-stage small-cell lung cancer.

To attempt to improve the poor prognosis of extensive-stage small-cell lung cancer (SCLC) patients, we tried to administer late intensive combined modality therapy (LICMRX) to patients with good tumor regression after 12 weeks of conventional chemotherapy. Twenty-nine consecutive extensive-stage SCLC patients received 6 weeks of cyclophosphamide, methotrexate, and lomustine (CMC) induction therapy, followed by 6 weeks of vincristine, doxorubicin, and procarbazine (VAP). After restaging for assessment of tumor response, autologous bone marrow (ABM) was collected in patients in good medical condition with complete response (CR) or partial response (PR) and no tumor on marrow examination. LICMRX consisted of irradiation with 2,000 rad in five fractions for five days to sites of initial tumor involvement, followed by cyclophosphamide, 60 mg/kg for 2 days, and etoposide, 200 mg/m2 for 3 days and then by ABM infusion. Prophylactic cranial irradiation (PCI) was administered thereafter, but no further chemotherapy was used. Due to lack of tumor regression or poor medical condition, only ten of the original 29 patients were eligible for LICMRX; two refused, so only eight (28%) received therapy. Three patients who began LICMRX in CR developed recurrence of SCLC after an additional 4, 8, and 15 months. Of five patients with PR, one attained CR but relapsed at 3 months, two remained in PR and progressed at 2 and 4 months, and two died of infection without recovery from LICMRX. Mean time from ABM infusion to recovery of granulocyte count to 500/microL was 15.8 days in the six surviving patients (range, 12-22). The major non-hematologic toxicity of LICMRX was severe esophagitis. Among all 29 patients, there were six CRs (21%) and no 2-year survivors, compared with a CR rate of 36% and 10% 2-year survivors in 78 extensive-stage patients previously treated with CMC plus VAP without LICMRX. We conclude that the LICMRX given in this study can be administered to only a minority of extensive-stage SCLC patients and is very unlikely to yield substantial improvement in the fraction of 2-year survivors (95% confidence limits for 2-year survival 0% to 10%).