Deciding whether a missense allelic variant affects protein function is important in many contexts. We previously demonstrated that a detailed analysis of p53 intragenic conservation correlates with somatic mutation hotspots. Here we refine these evolutionary studies and expand them to the p16/Ink4a gene. We calculated that in order for 'absolute conservation' of a codon across multiple species to achieve Po0.05, the evolutionary substitution database must contain at least 3(M) variants, where M equals the number of codons in the gene. Codons in p53 were divided into high (73% of codons), intermediate (29% of codons), and low (0 codons) likelihood of being mutation hotspots. From a database of 263 somatic missense p16 mutations, we identified only four codons that are mutational hotspots at Po0.05 (>8 mutations). However, data on function, structure, and disease association support the conclusion that 11 other codons with !5 somatic mutations also likely indicate functionally critical residues, even though P>0.05. We calculated p16 evolution using amino acid substitution matrices and nucleotide substitution distances. We looked for evolutionary parameters at each codon that would predict whether missense mutations were disease associated or disrupted function. The current p16 evolutionary substitution database is too small to determine whether observations of 'absolute conservation' are statistically significant. Increasing the number of sequences from three to seven significantly improved the predictive value of evolutionary computations. The sensitivity and specificity for conservation scores in predicting disease association of p16 codons is 70-80%. Despite the small p16 sequence database, our calculations of high conservation correctly predicted loss of cell cycle arrest function in 75% of tested codons, and low conservation correctly predicted wild-type function in 80-90% of codons. These data validate our hypothesis that detailed evolutionary analyses help predict the consequences of missense amino-acid variants.

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Purpose: The purpose of this study was to evaluate the prognostic effect of early posttransplant lymphocyte recovery in patients with advanced breast cancer receiving highdose chemotherapy with autologous hematopoietic progenitor cell transplantation.Experimental Design: We analyzed the effect of the absolute lymphocyte count on day ؉15 posttransplant on freedom from relapse and overall survival in patients with high-risk primary breast cancer or metastatic breast cancer, enrolled between 1990 and 2001 in prospective high-dose chemotherapy trials, using a uniform regimen of cyclophosphamide, cisplatin, and 1,3-bis(2-chloroethyl)-1-nitrosourea.Results: Four hundred and seventy-six patients (264 high-risk primary breast cancer and 212 metastatic breast cancer patients) were evaluated at median follow-up of 8 years (range, 1.5-11 years). The disease-free survival and overall survival rates in the high-risk primary breast cancer group were 67% and 70%, respectively. Patients with metastatic breast cancer patients had 21.8% disease-free survival and 31.5% overall survival rates. Day ؉15 absolute lymphocyte count correlated with freedom from relapse (P ‫؍‬ 0.007) and overall survival (P ‫؍‬ 0.04) in the metastatic breast cancer group, but not in the high-risk primary breast cancer group (P ‫؍‬ 0.5 and 0.8, respectively). The prognostic effect of absolute lymphocyte count in metastatic breast cancer was restricted to those patients receiving unmanipulated peripheral blood progenitor cells (P ‫؍‬ 0.04). In contrast, absolute lymphocyte count had no significant effect in those metastatic breast cancer patients receiving bone marrow or a CD34-selected product. In multivariate analyses, the prognostic effect of day ؉15 absolute lymphocyte count in metastatic breast cancer was independent of other predictors, such as disease status, pre-high-dose chemotherapy treatment, number of tumor sites, or HER2.Conclusions: Early lymphocyte recovery is an independent outcome predictor in metastatic breast cancer patients receiving high-dose chemotherapy and an autologous peripheral blood progenitor cell transplant. These observations suggest that immune strategies targeting minimal posttransplant residual disease may prove worthwhile.

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