Clinical radiation pneumonitis and radiographic changes after thoracic radiation therapy for lung carcinoma.

BACKGROUND: The authors attempted to determine the incidence of and risk factors for clinical radiation pneumonitis in patients treated for lung carcinoma. They also sought to describe the corresponding posttreatment radiographic changes. METHODS: Between 1989-1993, 83 patients received curative radiation therapy for lung carcinoma. Of these, 39 patients were treated with definitive radiation therapy, and 44 patients were treated with adjuvant radiation therapy after surgical resection. The median radiation therapy dose was 54 gray (Gy), and the median treatment area was 182 cm2. Chest radiographs obtained after radiation therapy were reviewed and scored for margin definition, volume loss, and texture quality. RESULTS: A total of 17 patients (20%) developed clinical radiation pneumonitis (CRP). The median radiation therapy dose for the CRP cohort was 54 Gy, and the median treatment volume was 193 cm2. The median time to onset of symptoms was 3 weeks after radiation therapy, and the median duration of symptoms was 10 weeks. Of the 15 evaluable patients, symptoms resolved for 9 patients, improved but persisted for 4 patients, and CRP was fatal for 2 patients. The incidence of CRP was increased for patients with low performance status, comorbid lung disease, smoking history, low pulmonary function tests, and for those patients who did not undergo a surgical resection. Posttreatment radiographic changes were common and progressed with time. Radiographic changes were more pronounced in the CRP cohort, and extended outside the radiation therapy treatment field in the majority of patients (67%). CONCLUSIONS: Clinical radiation pneumonitis developed in 20% of lung carcinoma patients. Risk factors included low performance status, comorbid lung disease, smoking history, low pulmonary function tests, and the absence of a surgical resection. Posttreatment radiograph changes were common and progressed with time, and typically were not confined to the radiation therapy treatment field.

Skin recurrences after breast-conserving therapy for early-stage breast cancer.

PURPOSE: To assess the frequency and prognosis of skin recurrences after breast-conserving therapy (BCT) compared with other breast recurrences. MATERIALS AND METHODS: From 1968 to 1986, 1,624 patients with unilateral stage I or II breast cancer treated with BCT at the Joint Center for Radiation Therapy (Boston, MA) underwent gross tumor excision and received a dose of > or = 60 Gy to the tumor bed. Skin recurrences (SR) were defined as breast recurrences without associated parenchymal disease. An invasive breast recurrence with any parenchymal disease noted clinically or radiographically was scored as an other breast recurrence (OBR). Median follow-up for survivors was 137 months. RESULTS: SR represented 8% (18 of 229) of all breast recurrences and occurred in 1.1% of all patients. The outcome after local recurrence was different for patients with SR and invasive OBR. Patients with SR more frequently had uncontrolled local failure (50%; 9 of 18) than did patients with OBR (14%; 26 of 188) (P = .0007). Forty-four percent (8 of 18) of patients with SR had distant metastasis simultaneously or within 2 months of the recurrence compared with 5% (9 of 188) of invasive OBR patients (P < .0001). For patients without distant metastasis at the time of recurrence, the 5-year actuarial rate of development of distant metastasis was 60% for SR patients compared with 39% for invasive OBR patients (P = .07), and the corresponding 5-year actuarial survival rates beyond the time of local failure were 51% and 79%, respectively (P = .06). CONCLUSION: In contrast to other types of invasive breast recurrence after breast-conserving therapy, skin recurrences are rare and are associated with a significantly higher rate of distant metastasis and uncontrolled local disease as well as a lower rate of survival.

Is machine energy (4-8 MV) associated with outcome for stage I-II breast cancer patients?

PURPOSE: To assess the relationship between machine energy (4-8 MV) and treatment outcome in patients treated with conservative surgery and radiation therapy. METHODS AND MATERIALS: Between 1968 and 1985, 1624 patients were treated for clinical Stage I or II invasive breast cancer. The study population was limited to 1380 patients who underwent complete gross excision and received greater than or equal to 60 Gy to the tumor bed. Of these, 1125 were treated on a 4 MV, 153 on a 6 MV, and 102 on an 8 MV linear accelerator. Patients were selected for treatment on the 8 MV machine based on chest wall separations greater than 24 cm. Of patients treated on the 8 MV, netting was used for 42% and bolus was used for 26%. The median dose with bolus was 14 Gy in seven fractions (range: 2-34.2 Gy). Patients treated on the 8 MV accelerator were older, had a higher percentage of clinical T2 tumors, a higher percentage of pathologically positive nodes, and a lower incidence of extensive intraductal component (EIC). Median follow-up times were 130, 153, and 102 months, respectively, for survivors treated on the 4, 6, and 8 MV machines. RESULTS: We analyzed the site and 5-year crude incidence of first failure by machine energy and found the pattern of first failure site (local, nodal, or distant) to be virtually identical for each energy group. Of the local failures, 12 were in the skin of the treated breast, and these failures were evenly distributed by machine energy. We performed a multivariate analysis to adjust for factors known to predict for treatment failure. When adjusted for these other variables, machine energy was not associated with an increased (or decreased) risk of recurrence (RR for 8 MV vs. 4 MV = 0.94, p = 0.7; RR for 6 MV vs. 4 MV = 1.0, p = 0.9). We also analyzed the nature and incidence of treatment complications (rib fracture, radiation pneumonitis, soft tissue necrosis, and brachial plexopathy) and found no significant differences among the three treatment groups when stratified by treatment technique (tangents only vs. three-field). There was also no significant difference in cosmetic outcome at 5 years among the three groups. CONCLUSIONS: We conclude that machine energy over the range of 4 to 8 MV does not significantly affect treatment outcome. Specifically, it was feasible to treat patients with large chest wall separations using an 8 MV machine without an increase in skin recurrences and with the improved dose homogeneity afforded by 8 MV machines as compared with those of lower energies.

Correlation of heat resistance and HSP-70A mRNA levels in human tumor cells measured by competitive quantitative polymerase chain reaction.

PURPOSE: Several HSP-70 genes have been cloned and sequenced in human cells. Among these genes, the HSP-70A mRNA and protein levels correlate best with the development and decay of thermotolerance and intrinsic thermal sensitivity. Leukemic and nonleukemic human tumor cells express low levels of the normally heat inducible HSP-70A mRNA in control nonheated cells. Using a competitive quantitative polymerase chain reaction, we have measured the levels of mRNA for this gene and have correlated it with both transient and intrinsic thermal sensitivity of tumor cells. Such studies were also extended to tumor samples obtained from patients. METHODS AND MATERIALS: In these studies, the plasmid phHSP-70 which contains the entire human HSP-70A gene was modified by the insertion of the T7 promoter at the 5'-end untranslated region as well as the insertion of a 23 bp synthetic linker at the BamH1 site in the promoter region of the HSP-70A gene. The PCR primers were located such that the amplified fragment contained the linker. Using the T7 polymerase, the HSP-70A mRNA was transcribed from this plasmid (phHSP-70L) in vitro. A known amount of HSP-70A mRNA was then added to the total RNA prepared from the cell samples or from the tumor tissues obtained from patients. Using the components of the PCR reaction plus known amounts of HSP-70A mRNA synthesized from phHSP-70L and unknown amounts of total cellular RNA, the samples were amplified and analysed on a denaturing acrylamide gel. The PCR products obtained from phHSP-70L were 23 bp larger than the PCR products obtained from the cell samples due to the addition of the synthetic linker to the HSP-70A gene in phHSP-70L and therefore, the two products could be easily distinguished from each other and quantitated. The alpha-32P-dCTP incorporated in each sample was quantitated by AMBIS Scanner. When the 32P-counts were equal in the known and the unknown samples, the amount of the HSP-70A mRNA was taken to be equal in the known and the unknown sample. RESULTS: The results show that HSP-70A mRNA levels can be used to predict the survival levels during the development and decay of thermotolerance. In nonleukemic human tumor cell lines, there are as much as 40-50-fold induction of HSP-70A mRNA levels during the peak of thermotolerance. In leukemic cell lines, however, HSP-70A mRNA levels are induced only by three-fold during the same time period. These differences between the levels of HSP-70A mRNA positively correlate with the amount of tolerance development in leukemic and nonleukemic tumor cells. HSP-70A mRNA levels also vary in different tumor cells under nonheated conditions and there is a positive correlation between HSP-70A mRNA levels in nonleukemic human tumor cells and the level of their intrinsic thermal resistance. CONCLUSION: HSP-70A mRNA levels can be used to predict the intrinsic thermal sensitivity of nonleukemic human tumor cells.

Expression of HSP-28 and three HSP-70 genes during the development and decay of thermotolerance in leukemic and nonleukemic human tumors.

Leukemic cells appear to develop less thermotolerance and then to lose their thermotolerance more rapidly than do other tumor cell lines. The reason for this phenomenon is not known. After heat shock (or other environmental stresses), mammalian cells preferentially synthesize a set of proteins known as heat shock proteins (HSPs). HSP-28 and the various isoforms of HSP-70 have been suggested as being responsible for the development of thermotolerance. In these studies, we have attempted to determine by their expression with HSPs positively correlate with the development and decay of thermotolerance and whether the expression of these genes could explain the differing thermotolerance response observed between leukemic and nonleukemic tumor cells. Polymerase chain reaction was used to detect the expression of HSP-28 and several HSP-70 genes. Our data indicate that the expression of all three heat-inducible HSP-70 genes, 70A (Hunt and Morimoto, Proc. Natl. Acad. Sci. USA, 82: 6455-6459, 1985), 70B (Voellmy et al., Proc. Natl. Acad. Sci. USA, 82: 4949-4953, 1985), and 70B' (Leung et al., Biochem J., 267: 125-132, 1990) correlate with the development and decay of thermotolerance in nonleukemic tumor cell lines after heat or arsenite treatment. HSP-28 (Hickey et al., Nucleic Acids Res., 4: 4127-4145, 1986) failed to correlate with thermotolerance development; it was not induced after 45 degrees C primary heat shock. In leukemic cells, however, none of the HSPs were induced for extended periods of time. The lack of coordinate expression of HSP genes in cells of myeloid origin may explain the poor induction and maintenance of thermotolerance that is observed in these cells.

Effective vaccination of mice against leprosy bacilli with subunits of Mycobacterium leprae.

Model vaccines against leprosy bacilli have consisted of nonvirulent, live, attenuated Mycobacterium bovis BCG and irradiated, heat-killed, or autoclaved intact M. leprae. We report that immunization with various cell wall fractions of M. leprae, progressively depleted of lipids, carbohydrates, and soluble proteins, as well as a partially purified protein(s) derived from a pellet fraction of sonicated M. leprae, conferred significant protection against subsequent infection with live leprosy bacilli. Moreover, lymphocytes from regional lymph nodes and spleens of mice immunized with these M. leprae-derived subunits responded by proliferation when stimulated with M. leprae in vitro. Our results provide the first evidence that vaccination with M. leprae-derived fractions protects mice against leprosy bacilli.

Basement membrane procollagen IV and its specialized carboxyl domain are conserved in Drosophila, mouse, and human.

Interaction with the extracellular matrix is important for the proliferation and differentiation of cells during development. A specialized extracellular matrix, basement membrane, is built around a scaffold of procollagen IV molecules. We report the sequence of a 2.5-kilobase cDNA which contains the carboxyl end of a Drosophila melanogaster procollagen IV. The amino acid sequence of the carboxyl-terminal domain, which forms an essential intermolecular linkage between procollagen IV molecules, is 59% identical in Drosophila and vertebrate procollagens IV, and an additional 17% of residues are conservatively substituted. This implies that the nature of the linkage is also conserved. We suggest that intermolecular junctions through procollagen IV carboxyl domains are fundamental elements of the molecular architecture of Metazoan basement membranes and have been conserved during evolution. The isolation and identification of this basement membrane collagen gene of Drosophila will help in deducing the function of procollagen IV in basement membranes.

Assembly of procollagen mRNA translation products into pepsin-resistant structures.

Proteolytic digestion was used to probe the conformation of the prepro alpha chains synthesized by a mRNA-dependent reticulocyte lysate from chicken calvarial RNA. Pepsin-resistant alpha 1- and alpha 2-like chains were recovered even from translation reactions that were not preincubated below the reported Tm of the unhydroxylated triple helix. The pepsin-resistant structures were stable to thermal denaturation at 45 degrees C and a fraction remained resistant to peptic digestion at 30 degrees C. Interchain disulfide bonds did not appear to be required for the formation or thermal stability of these structures. Pepsin resistance is normally interpreted as evidence for a triple-helical conformation. Therefore, these results suggest that the in vitro synthesized prepro alpha chains contain the requisite information to associate in register for correct helix folding. The unusual thermal stability of these structures is not understood, but this may indicate assembly into higher orders of structure.

DNA sequence analysis of a mouse pro alpha 1 (I) procollagen gene: evidence for a mouse B1 element within the gene.

In a 3.8-kilobase mouse DNA sequence encoding amino acid sequences for the pro alpha 1(I) chain of type I procollagen, 14 coding sequences were identified which specify a sequence 95% homologous to amino acid residues 568 to 963 of the bovine alpha 1(I) chain. All of these coding sequences were flanked by appropriate splice junctions following the GT/AG rule. These observations suggest, but do not prove, that this pro alpha 1(I) gene is transcriptionally active. Of the 14 coding sequences, 7 were 54 base pairs in length, whereas the remainder were higher multiples of 54 base pairs. Nonrandom utilization of codons pertained throughout all of the coding sequences showing a preference (56%) for U in the wobble position. Two of the intervening sequences encoded imperfect vestiges of coding sequences which exhibited a codon preference different from that of the pro alpha 1(I) gene proper and were not flanked by splice junctions. One intervening sequence encoded a member of the mouse B1 family of middle repetitive sequences. It was flanked by 8-base-pair direct repeats and had a truncated A-rich region, suggesting that it may be a mobile element. Within this element were sequences which could function as a RNA polymerase III split promoter.

Expression and novel structure of a collagen gene in Drosophila.

We report the structure and developmental expression of collagen gene sequences in Drosophila melanogaster. Collagen-like genomic clones were isolated by screening a Drosophila genomic library with a chicken pro alpha 2(I) cDNA clone as a hybridization probe. A 1.5-kilobase (kb) DNA sequence from a 9.2-kb DNA clone (pDCg1) is presented. Unlike the highly fragmented genes for vertebrate type I collagen, there is no evidence of a 54-base-pair primordial unit within this gene segment. Instead, the fragment is composed of two large coding sequences. Together they specify a sequence of 469 amino acids. This collagen product is composed almost entirely of the Gly-X-Y repeat characteristic of peptides involved in triple helix formation. Within the polypeptide there are four minor discontinuities in the Gly-X-Y pattern. Similar interruptions have been observed in a mouse basement membrane collagen protein sequence. Therefore, the Drosophila collagen gene may encode a nonfibrous collagen such as a basement membrane or cuticle collagen or a novel collagenous protein. By using the DNA segment of known sequence as a hybridization probe, a developmental sequence of polyadenylylated RNA samples was screened for the presence of homologous sequences. A RNA species 6.4 kb in length was detected as a prominent band only in the first- and second-instar larval stages. This pattern of developmental hybridization correlates with the production of the cuticle and basement membranes, and the large size of the RNA is consistent with its identification as a collagen-encoding RNA.

Comparison of the NH2-Terminal sequences of chick Type I preprocollagen chains synthesized in an nRNA-dependent reticulocyte lysate.

RNA was extracted from 17-day-old chick embryo calvaria and translated by an mRNA-dependent reticulocyte lysate. Procedures were developed for purifying intact pro alpha 1(I) and pro alpha 2 translation products from the lysate. These products were identified by comparing tryptic peptide elution patterns of the translation products with those obtained from secreted pro alpha chains. Partial sequence data from the amino terminus of each translation product demonstrated that each chain begins with a sequence that is different from that of the corresponding pro alpha chain, and that the two sequences are not the same. Also, the bacterial collagenase-resistant peptide from the amino terminus of prepro alpha 1(I) had an apparent molecular weight which was 10 000 greater than the peptide from pro alpha 1(I).

Identification of a Balb/c mouse pro alpha 1(I) procollagen gene: evidence for insertions or deletions in gene coding sequences.

We report the first isolation and identification of a mouse genomic fragment encoding amino acid sequences for the pro alpha 1(I) chain of type I procollagen. The DNA sequence of eight coding sequences is presented; five of these are 54 bp and three are 108 bp in length. Together these specify 198 amino acids which are 94% homologous to the corresponding bovine pro alpha 1(I) chain protein sequences. Each of the eight coding sequences is flanked by appropriate splice-junction sequences that exhibit considerable sequence complementarity to the rat small nuclear U1a RNA. In the 198 codons examined in this mouse genomic clone, the preferred codons for glycine and alanine are GGU (46/67) and GCU (23/30), respectively. This is in contrast to the codon usage reported for the chicken pro alpha 1(I) cDNA clone (Fuller and Boedtker, 1981). The examined coding sequences exhibit considerable nucleotide homology in both end-to-end and in staggered alignments. Based on an analysis of this homology data, a model is presented for the generation of 108-bp coding sequences from 54-bp units by two successive homologous recombinational events within coding sequences. Alternatively, the 108-bp units may have arisen by precise deletions of an intervening sequence between 54-bp coding sequences. Evidence supporting this is provided by a comparison of pro alpha 1(I) and pro alpha 2(I) genes. In the mouse pro alpha 1(I) gene amino acids 856-891 are encoded in a 108-bp unit; the chicken pro alpha 2(I) gene these residues are encoded in two 54-bp coding sequences. In addition, the coding sequences for nearly 50% of the alpha domain are condensed in the pro alpha 1(I) gene into a region approximately one half the size occupied by the comparable sequences in the pro alpha 2(I) gene.

Translation of chick calvarial procollagen messenger RNA'S by a messenger RNA dependent reticulocyte lysate.

A method was developed for the extraction of RNA from chick embryo calvaria which should be generally applicable to other connective tissues. Total RNA prepared by this method was translated by a mRNA-dependent reticulocyte lysate into discrete pro alpha chains. Several criteria were used to identify these translation products, including (1) preferential labeling with [3H]proline, (2) appropriate migration on sodium dodecyl sulfate-polyacrylamide gels, (3) selective sensitivity to collagenase digestion, and (4) specific precipitability by two different antisera against procollagen. Data from the immunoprecipitation experiments indicated that the majority of the pro alpha chains contained the carboxy-terminal antigenic determinants. These results demonstrate that this translation system can be used as an assay for intact procollagen mRNAs and as a source of in vitro synthesized pro alpha chains for future structural analysis.

Identification of a disulfide-linked procollagen as the biosynthetic precursor of chick-bone collagen.

Chick cranial-bone procollagen, extracted at neutral pH in the presence of inhibitors of proteolytic enzymes, exists as a triple-stranded protein with disulfide bonds linking all three chains. The biosynthetic precursor function of this procollagen was demonstrated by pulsechase experiments. The ratio of radioactive hydroxyproline to proline in proalpha chains obtained by reduction and alkylation of the disulfide-bonded precursor was similar to the value determined for proalpha1 from acid-extracted procollagen. However, the molecular weight of these chains was higher than that previously determined for proalpha1 and proalpha2, suggesting that extraction of tissue at low pH results in a selective loss of disulfide-bonded regions from procollagen. These findings reconcile several apparently conflicting reports on the nature of procollagen identified in bone and in the medium of cultured fibroblasts and indicate that in both systems procollagen is synthesized as a disulfidelinked protein.