Maria J. Merino

Activating mutations of the stimulatory G protein in the McCune-Albright syndrome.

BACKGROUND The McCune-Albright syndrome is a sporadic disease characterized by polyostotic fibrous dysplasia, café au lait spots, sexual precocity, and hyperfunction of multiple endocrine glands. These manifestations may be explained by a somatic mutation in affected tissues that results in activation of the signal-transduction pathway generating cyclic AMP (cAMP). We analyzed DNA from tissues of patients with the McCune-Albright syndrome for the presence of activating mutations of the gene for the alpha subunit of the G protein (Gs alpha) that stimulates cAMP formation. METHODS Genomic DNA fragments encompassing regions (exons 8 and 9) previously found to contain activating missense mutations of the Gs alpha gene (gsp mutations) in sporadically occurring pituitary tumors were amplified in tissues from four patients with the McCune-Albright syndrome by the polymerase chain reaction. The amplified DNA was analyzed for mutations by denaturing gradient gel electrophoresis and allele-specific oligonucleotide hybridization. RESULTS We detected one of two activating mutations within exon 8 of the Gs alpha gene in tissues from all four patients, including affected endocrine organs (gonads, adrenal glands, thyroid, and pituitary) and tissues not classically involved in the McCune-Albright syndrome. In two of the patients, histidine was substituted for arginine at position 201 of Gs alpha, and in the other two patients cysteine was substituted for the same arginine residue. In each patient the proportion of cells affected varied from tissue to tissue. In two endocrine organs, the highest proportion of mutant alleles was found in regions of abnormal cell proliferation. CONCLUSIONS Mutations within exon 8 of the Gs alpha gene that result in increased activity of the Gs protein and increased cAMP formation are present in various tissues of patients with the McCune-Albright syndrome. Somatic mutation of this gene early in embryogenesis could result in the mosaic population of normal and mutant-bearing tissues that may underlie the clinical manifestations of this disease.

Gene transfer into humans--immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction.

BACKGROUND AND METHODS Treatment with tumor-infiltrating lymphocytes (TIL) plus interleukin-2 can mediate the regression of metastatic melanoma in approximately half of patients. To optimize this treatment approach and define the in vivo distribution and survival of TIL, we used retroviral-mediated gene transduction to introduce the gene coding for resistance to neomycin into human TIL before their infusion into patients--thus using the new gene as a marker for the infused cells. RESULTS Five patients received the gene-modified TIL. All the patients tolerated the treatment well, and no side effects due to the gene transduction were noted. The presence and expression of the neomycin-resistance gene were demonstrated in TIL from all the patients with Southern blot analysis and enzymatic assay for the neomycin phosphotransferase coded by the bacterial gene. Cells from four of the five patients grew successfully in high concentrations of G418, a neomycin analogue otherwise toxic to eukaryotic cells. With polymerase-chain-reaction analysis, gene-modified cells were consistently found in the circulation of all five patients for three weeks and for as long as two months in two patients. Cells were recovered from tumor deposits as much as 64 days after cell administration. The procedure was safe according to all criteria, including the absence of infections virus in TIL and in the patients. CONCLUSIONS These studies demonstrate the feasibility and safety of using retroviral gene transduction for human gene therapy and have implications for the design of TIL with improved antitumor potency, as well as for the possible use of lymphocytes for the gene therapy of other diseases.

Randomized prospective study of the benefit of adjuvant radiation therapy in the treatment of soft tissue sarcomas of the extremity.

PURPOSE This randomized, prospective study assesses the impact of postoperative external-beam radiation therapy on local recurrence (LR), overall survival (OS), and quality of life after limb-sparing resection of extremity sarcomas. PATIENTS AND METHODS Patients with extremity tumors and a limb-sparing surgical option were randomized to receive or not receive postoperative adjuvant external-beam radiotherapy. Patients with high-grade sarcomas received postoperative adjuvant chemotherapy whereas patients with low-grade sarcomas or locally aggressive nonmalignant tumors were randomized after surgery alone. RESULTS Ninety-one patients with high-grade lesions were randomized; 47 to receive radiotherapy (XRT) and 44 to not receive XRT. With a median follow-up of 9.6 years, a highly significant decrease (P2 = .0028) in the probability of LR was seen with radiation, but no difference in OS was shown. Of 50 patients with low-grade lesions (24 randomized to resection alone and 26 to resection and postoperative XRT), there was also a lower probability of LR (P2 = .016) in patients receiving XRT, again, without a difference in OS. A concurrent quality-of-life study showed that extremity radiotherapy resulted in significantly worse limb strength, edema, and range of motion, but these deficits were often transient and had few measurable effects on activities of daily life or global quality of life. CONCLUSION This study indicates that although postoperative external-beam radiotherapy is highly effective in preventing LRs, selected patients with extremity soft tissue sarcoma who have a low risk of LR may not require adjuvant XRT after limb-sparing surgery (LSS).

Randomized Study of High-Dose and Low-Dose Interleukin-2 in Patients With Metastatic Renal Cancer

PURPOSE This three-arm randomized study compares response rates and overall survival of patients with metastatic renal cell cancer (RCC) receiving high-dose or one of two low-dose interleukin-2 (IL-2) regimens. PATIENTS AND METHODS Patients with measurable metastatic RCC and a good performance status were randomized to receive either 720,000 U/kg (high-dose [HD]) or 72,000 U/kg (low-dose [LD]), both given by intravenous (IV) bolus every 8 hours. After randomly assigning 117 patients, a third arm of low-dose daily subcutaneous IL-2 was added, and an additional 283 patients were randomly assigned. RESULTS A total of 156 patients were randomly assigned to HD IV IL-2, and 150 patients to LD IV IL-2. Toxicities were less frequent with LD IV IL-2 (especially hypotension), but there were no IL-2-related deaths in any arm. There was a higher response proportion with HD IV IL-2 (21%) versus LD IV IL-2 (13%; P =.048) but no overall survival difference. The response rate of subcutaneous IL-2 (10%, partial response and complete response) was similar to that of LD IV IL-2, differing from HD IV (P =.033). Response durability and survival in completely responding patients was superior with HD IV compared with LD IV therapy (P =.04). CONCLUSION Major tumor regressions, as well as complete responses, were seen with all regimens tested. IL-2 was more clinically active at maximal doses, although this did not produce an overall survival benefit. The immunological factors which constrain the curative potential of IL-2 to only a small percentage of patients need to be further elucidated.

Mutations in a novel gene lead to kidney tumors, lung wall defects, and benign tumors of the hair follicle in patients with the Birt-Hogg-Dubé syndrome

Birt-Hogg-Dubé (BHD) syndrome is a rare inherited genodermatosis characterized by hair follicle hamartomas, kidney tumors, and spontaneous pneumothorax. Recombination mapping in BHD families delineated the susceptibility locus to 700 kb on chromosome 17p11.2. Protein-truncating mutations were identified in a novel candidate gene in a panel of BHD families, with a 44% frequency of insertion/deletion mutations within a hypermutable C(8) tract. Tissue expression of the 3.8 kb transcript was widespread, including kidney, lung, and skin. The full-length BHD sequence predicted a novel protein, folliculin, that was highly conserved across species. Discovery of disease-causing mutations in BHD, a novel kidney cancer gene associated with renal oncocytoma or chromophobe renal cancer, will contribute to understanding the role of folliculin in pathways common to skin, lung, and kidney development.

gp100 Peptide Vaccine and Interleukin-2 in Patients with Advanced Melanoma

BACKGROUND Stimulating an immune response against cancer with the use of vaccines remains a challenge. We hypothesized that combining a melanoma vaccine with interleukin-2, an immune activating agent, could improve outcomes. In a previous phase 2 study, patients with metastatic melanoma receiving high-dose interleukin-2 plus the gp100:209-217(210M) peptide vaccine had a higher rate of response than the rate that is expected among patients who are treated with interleukin-2 alone. METHODS We conducted a randomized, phase 3 trial involving 185 patients at 21 centers. Eligibility criteria included stage IV or locally advanced stage III cutaneous melanoma, expression of HLA*A0201, an absence of brain metastases, and suitability for high-dose interleukin-2 therapy. Patients were randomly assigned to receive interleukin-2 alone (720,000 IU per kilogram of body weight per dose) or gp100:209-217(210M) plus incomplete Freunds adjuvant (Montanide ISA-51) once per cycle, followed by interleukin-2. The primary end point was clinical response. Secondary end points included toxic effects and progression-free survival. RESULTS The treatment groups were well balanced with respect to baseline characteristics and received a similar amount of interleukin-2 per cycle. The toxic effects were consistent with those expected with interleukin-2 therapy. The vaccine-interleukin-2 group, as compared with the interleukin-2-only group, had a significant improvement in centrally verified overall clinical response (16% vs. 6%, P=0.03), as well as longer progression-free survival (2.2 months; 95% confidence interval [CI], 1.7 to 3.9 vs. 1.6 months; 95% CI, 1.5 to 1.8; P=0.008). The median overall survival was also longer in the vaccine-interleukin-2 group than in the interleukin-2-only group (17.8 months; 95% CI, 11.9 to 25.8 vs. 11.1 months; 95% CI, 8.7 to 16.3; P=0.06). CONCLUSIONS In patients with advanced melanoma, the response rate was higher and progression-free survival longer with vaccine and interleukin-2 than with interleukin-2 alone. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT00019682.).

T Cells Targeting Carcinoembryonic Antigen Can Mediate Regression of Metastatic Colorectal Cancer but Induce Severe Transient Colitis

Autologous T lymphocytes genetically engineered to express a murine T cell receptor (TCR) against human carcinoembryonic antigen (CEA) were administered to three patients with metastatic colorectal cancer refractory to standard treatments. All patients experienced profound decreases in serum CEA levels (74-99%), and one patient had an objective regression of cancer metastatic to the lung and liver. However, a severe transient inflammatory colitis that represented a dose limiting toxicity was induced in all three patients. This report represents the first example of objective regression of metastatic colorectal cancer mediated by adoptive T cell transfer and illustrates the successful use of a TCR, raised in human leukocyte antigen (HLA) transgenic mice, against a human tumor associated antigen. It also emphasizes the destructive power of small numbers of highly avid T cells and the limitations of using CEA as a target for cancer immunotherapy.Autologous T lymphocytes genetically engineered to express a murine T cell receptor (TCR) against human carcinoembryonic antigen (CEA) were administered to three patients with metastatic colorectal cancer refractory to standard treatments. All patients experienced profound decreases in serum CEA levels (74-99%), and one patient had an objective regression of cancer metastatic to the lung and liver. However, a severe transient inflammatory colitis that represented a dose limiting toxicity was induced in all three patients. This report represents the first example of objective regression of metastatic colorectal cancer mediated by adoptive T cell transfer and illustrates the successful use of a TCR, raised in human leukocyte antigen (HLA) transgenic mice, against a human tumor associated antigen. It also emphasizes the destructive power of small numbers of highly avid T cells and the limitations of using CEA as a target for cancer immunotherapy.

The mammary pathology of genetically engineered mice: the consensus report and recommendations from the Annapolis meeting{

NIH sponsored a meeting of medical and veterinary pathologists with mammary gland expertise in Annapolis in March 1999. Rapid development of mouse mammary models has accentuated the need for definitions of the mammary lesions in genetically engineered mice (GEM) and to assess their usefulness as models of human breast disease. The panel of nine pathologists independently reviewed material representing over 90% of the published systems. The GEM tumors were found to have: (1) phenotypes similar to those of non-GEM; (2) signature phenotypes specific to the transgene; and (3) some morphological similarities to the human disease. The current mouse mammary and human breast tumor classifications describe the majority of GEM lesions but unique morphologic lesions are found in many GEM. Since little information is available on the natural history of GEM lesions, a simple morphologic nomenclature is proposed that allows direct comparisons between models. Future progress requires rigorous application of guidelines covering pathologic examination of the mammary gland and the whole animal. Since the phenotype of the lesions is an essential component of their molecular pathology, funding agencies should adopt policies ensuring careful morphological evaluation of any funded research involving animal models. A pathologist should be part of each research team.

Mutations in the Fumarate Hydratase Gene Cause Hereditary Leiomyomatosis and Renal Cell Cancer in Families in North America

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an autosomal dominant disorder characterized by smooth-muscle tumors of the skin and uterus and/or renal cancer. Although the identification of germline mutations in the fumarate hydratase (FH) gene in European families supports it as the susceptibility gene for HLRCC, its role in families in North America has not been studied. We screened for germline mutations in FH in 35 families with cutaneous leiomyomas. Sequence analysis revealed mutations in FH in 31 families (89%). Twenty different mutations in FH were identified, of which 18 were novel. Of these 20 mutations, 2 were insertions, 5 were small deletions that caused frameshifts leading to premature truncation of the protein, and 13 were missense mutations. Eleven unrelated families shared a common mutation: R190H. Eighty-one individuals (47 women and 34 men) had cutaneous leiomyomas. Ninety-eight percent (46/47) of women with cutaneous leiomyomas also had uterine leiomyomas. Eighty-nine percent (41/46) of women with cutaneous and uterine leiomyomas had a total hysterectomy, 44% at age < or =30 years. We identified 13 individuals in 5 families with unilateral and solitary renal tumors. Seven individuals from four families had papillary type II renal cell carcinoma, and another individual from one of these families had collecting duct carcinoma of the kidney. The present study shows that mutations in FH are associated with HLRCC in North America. HLRCC is associated with clinically significant uterine fibroids and aggressive renal tumors. The present study also expands the histologic spectrum of renal tumors and FH mutations associated with HLRCC.

Renal tumors in the Birt-Hogg-Dubé syndrome.

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant genodermatosis characterized by the development of small dome-shaped papules on the face, neck, and upper trunk (fibrofolliculomas). In addition to these benign hair follicle tumors, BHD confers an increased risk of renal neoplasia and spontaneous pneumothorax. To date, there has been no systematic pathologic analysis of the renal tumors associated with this syndrome. We reviewed 130 solid renal tumors resected from 30 patients with BHD in 19 different families. Preoperative computed tomography scans demonstrated a mean of 5.3 tumors per patient (range 1–28 tumors), the largest tumors averaging 5.7 cm in diameter (± 3.4 cm, range 1.2–15 cm). Multiple and bilateral tumors were noted at an early age (mean 50.7 years). The resected tumors consisted predominantly of chromophobe renal cell carcinomas (44 of 130, 34%) or of hybrid oncocytic neoplasms that had areas reminiscent of chromophobe renal cell carcinoma and oncocytoma (65 of 130, 50%). Twelve clear cell (conventional) renal carcinomas (12 of 130, 9%) were diagnosed in nine patients. These tumors were on average larger (4.7 ± 4.2 cm) than the chromophobe (3.0 ± 2.5 cm) and hybrid tumors (2.2 ± 2.4 cm). Microscopic oncocytosis was found in the renal parenchyma of most patients, including the parenchyma of five patients with evidence of clear cell renal cell carcinoma. Our findings suggest that microscopic oncocytic lesions may be precursors of hybrid oncocytic tumors, chromophobe renal cell carcinomas, and perhaps clear cell renal cell carcinomas in patients with BHD syndrome. Recognition by the pathologist of the unusual renal tumors associated with BHD may assist in the clinical diagnosis of the syndrome.