Carl O'Hara

High-Dose Melphalan and Autologous Stem-Cell Transplantation in Patients with AL Amyloidosis: An 8-Year Study

Context AL amyloidosis responds poorly to oral chemotherapy and rarely leads to elimination of plasma cell dyscrasia. Amyloid cardiomyopathy is a particularly fatal complication of the disease. Contribution Analysis of consecutive patients with AL amyloidosis from 6 separate trials over 8 years shows that high-dose intravenous melphalan therapy combined with autologous stem-cell transplantation greatly improves duration of survival and ameliorates organ dysfunction. Implications Intravenous melphalan therapy combined with stem-cell transplantation represents a clinically significant improvement in treating AL amyloidosis and shows promise in reversing amyloid cardiomyopathy. The Editors The most common form of systemic amyloidosis in the United States is AL (or primary) amyloidosis. In this disease, amyloid fibrils are derived from monoclonal immunoglobulin light chains that are produced by an underlying clonal plasma cell dyscrasia. Although the burden of plasma cells is generally low, accumulation of amyloid deposits in vital organs leads to progressive disability and death. The median survival of untreated patients after diagnosis is 12 months and less than 5 months for those with cardiomyopathy (1-5). AL amyloidosis is reported to occur in 5 to 12 persons per million per year in the United States; however, death records and autopsy results suggest that the incidence may be higher (6, 7). Treatment with oral melphalan results in a modest increase in median survival but rarely eliminates the plasma cell dyscrasia and is not effective for rapidly progressive disease (8-10). Alternative chemotherapy regimens have not improved survival further (11-15). Promising treatment outcomes observed with high-dose intravenous melphalan and autologous stem-cell transplantation in multiple myeloma (16-19) provided a rationale for testing the hypothesis that this treatment would improve survival for patients with AL amyloidosis. Favorable responses to high-dose melphalan and stem-cell transplantation in patients with AL amyloidosis have been reported in case reports and in small series; however, treatment-related mortality was high in multicenter trials (20-28). Our initial experience with treatment in AL amyloidosis indicated that selected patients can tolerate treatment and that hematologic responses and reversal of amyloid-related organ dysfunction can be achieved (29-32). Since 1994, we have evaluated 701 patients with AL amyloidosis, 312 of whom initiated high-dose melphalan treatment and stem-cell transplantation. This longitudinal study examines survival, hematologic response, and improvement of amyloid-related organ disease in patients who were treated with high-dose melphalan and stem-cell transplantation. We contrast these data with features and survival of a simultaneous cohort of patients who were not eligible for treatment. Methods Patients Between July 1994 and June 2002, 701 consecutive patients with AL amyloidosis were evaluated and clinical data were collected with the approval of the Institutional Review Board of Boston University Medical Center. All patients had biopsy-proven amyloid disease and a documented plasma cell dyscrasia, which was diagnosed by the presence of clonal plasma cells in the bone marrow or a monoclonal gammopathy detected by immunofixation electrophoresis of serum or urine proteins (Figure 1). To exclude another type of systemic amyloidosis and a monoclonal gammopathy of unknown significance, all patients with findings compatible with familial or secondary (AA) amyloidosis were tested by DNA analysis for gene mutations in transthyretin, apolipoprotein A1, fibrinogen, and lysozyme known to be associated with amyloidosis and by immunohistochemistry of the biopsy tissue for AA amyloid fibril deposits (33). Patients with multiple myeloma (bone marrow plasmacytosis 30% or lytic bone lesions) were excluded. In patients older than 70 years of age with cardiomyopathy only, a diagnosis of senile cardiac amyloidosis (caused by wild-type transthyretin) was excluded by immunohistochemical examination of a tissue biopsy specimen using antiserum to transthyretin. All patients were evaluated for degree of organ involvement by physical examination, standardized blood tests, electrocardiography, echocardiography, chest radiography, pulmonary function tests, and a 24-hour urine collection. All patients were evaluated by a hematologist and cardiologist and, when appropriate, by nephrology, pulmonology, gastroenterology, and neurology specialists. Figure 1. Algorithm for patient selection and treatment with high-dose melphalan and stem-cell transplantation. High-Dose Melphalan and Stem-Cell Transplantation Eligibility and Protocols Patients were enrolled in several sequential institutional review boardapproved protocols during the 8-year study period. Eligibility criteria for all protocols required biopsy-proven amyloid disease; evidence of a plasma cell dyscrasia; at least 1 major organ affected by amyloid disease; and minimum measures of cardiac, pulmonary, and performance status (Figure 1). Functional measures included cardiac ejection fraction 0.4 or greater, absence of symptomatic pleural effusions, absence of heart failure or arrhythmia resistant to medical management, oxygen saturation of 95% or greater on room air, lung diffusing capacity of 50% or more of predicted, supine systolic blood pressure of 90 mm Hg or greater, and Southwest Oncology Group performance status score of 2 or less unless limited by neuropathy (on a scale of 0 to 4, reflecting percentage of the day [0%, 25%, 50%, 75%, or 100%] spent in bed or in a chair). Minor variations in eligibility requirements for age, renal function, amount of previous chemotherapy, and time from diagnosis while on some protocols are noted in the following discussion; the number of patients affected is also given. The first protocol (July 1994 to December 1995) enrolled 13 patients 60 years of age or younger with serum creatinine values of 176.8 mol/L (2.0 mg/dL) or less; these patients were treated with melphalan, 200 mg/m2 (29). Subsequent protocols had no restriction for impaired renal function. A second protocol (April 1995 to October 1996) enrolled 28 patients 70 years of age or younger and used a lower dose of melphalan, 100 mg/m2 (31). Two protocols (January 1996 to June 1998) evaluated the use of CD34+-selected stem cells in 16 patients (34). The fifth protocol (October 1996 to September 2000) randomly assigned 100 previously untreated patients to treatment with high-dose melphalan and stem-cell transplantation immediately or after 2 cycles of oral melphalan and prednisone. There was no age limit for this protocol; however, melphalan, 140 mg/m2, was given to patients who were older than 65 years of age or had a cardiac ejection fraction between 0.40 and 0.44. The sixth protocol (November 2000 to the present) has enrolled 29 patients 65 years of age or younger. On this protocol, enough stem cells are collected initially to give a second cycle of chemotherapy within the first year if a complete response has not been achieved after an initial course of melphalan at a dose of 200 mg/m2. Other patients who met eligibility criteria (August 1996 to the present) but were excluded from an active protocol because of previous treatment or time from diagnosis were treated by using the established dosing guidelines. Patients who did not meet eligibility for treatment with high-dose melphalan and stem-cell transplantation were grouped according to reasons for ineligibility and were analyzed for survival. Organ system involvement was defined by physical examination; postural blood pressure determinations; standardized serologic laboratory measurements of kidney, liver, and endocrine function; coagulation studies, including factor X levels; electrocardiography; echocardiography; chest radiography; pulmonary function tests with walking oximetry; and a 24-hour urine collection for protein excretion. Cardiac involvement was defined by septal or posterior wall thickening of 13 mm or greater on echocardiography or a clinical syndrome of congestive heart failure or cardiac arrhythmia in the absence of preexisting cardiac disease. Renal involvement was diagnosed by proteinuria of 500 mg/24 h or greater or an elevated serum creatinine concentration in the absence of other causes of renal disease. Gastrointestinal involvement was diagnosed by involuntary loss of 10% of body weight, unexplained diarrhea, hepatomegaly of 4 cm or more below the right costal margin on physical examination, or alkaline phosphatase level 2 or more times the upper limit of normal values. Peripheral neuropathy was diagnosed by symptoms and physical examination or nerve conduction studies, and autonomic neuropathy was defined by orthostatic hypotensiona decrease in systolic blood pressure of 20 mm Hg or greater with upright posture in euvolemic patients. Soft tissue involvement was diagnosed by clinical evidence of macroglossia, soft tissue or subcutaneous deposits, amyloid arthropathy, lymphadenopathy, or nail dystrophy. Coagulation factor X level was considered deficient if it was 50% or less of normal. Stem-Cell Collection and High-Dose Chemotherapy Peripheral blood stem cells were collected by leukapheresis after mobilization using granulocyte colony-stimulating factor. A minimum yield of 2.0 106 CD34+cells/kg of body weight was required to support high-dose chemotherapy. The patients age and cardiac status and the number of stem cells collected determined the melphalan dose (Figure 1). A dose of 200 mg/m2 was administered to patients who were 65 years of age or younger and who had a cardiac ejection fraction of 0.45 or greater and a stem-cell collection of at least 2.5 106 CD34+cells/kg. A dose of 140 mg/m2 was administered to patients who were older than 65 years of age, who had a cardiac ejection fraction of 0.4 to 0.44, or who had a stem-cell collection of 2.0 to 2.5 106 CD34+cells/k

Cardiac amyloidosis in African Americans: Comparison of clinical and laboratory features of transthyretin V122I amyloidosis and immunoglobulin light chain amyloidosis

BACKGROUND Transthyretin (TTR) mutations known to cause cardiac amyloidosis include V122I, found almost exclusively in African Americans at a prevalence of 3-3.9%. This retrospective study describes TTR V122I-associated cardiac amyloid disease (ATTR) in a major amyloid referral clinic population. METHODS Self-identified African Americans with amyloidosis (n = 156) were screened for TTR V122I by serum isoelectric focusing; mutant TTR was confirmed by DNA sequencing or mass spectrometry. Cardiac findings in ATTR V122I and immunoglobulin light chain (AL) amyloidoses were compared. RESULTS TTR V122I was identified in 36/156 (23.1%) of evaluated patients and included 5 homozygotes; the allele frequency was 0.013. One compound heterozygote (F44L/V122I) and 4 patients who had AL and the mutant TTR allele were characterized. In patients negative for V122I, AL was the most frequent diagnosis (86/120). Cardiomyopathy was present in 100% of patients with ATTR and 84% of patients with AL (P = .01). In patients with dominant cardiac involvement, better survival occurred in ATTR (n = 30) compared to AL (n = 31), (27 vs 5 months, P < .01) although the mean age in ATTR was higher (70.3 vs 56.2 years, P < .01). Congestive heart failure symptoms and electrocardiographic findings were similar in ATTR and AL, but significant differences in echocardiographic measurements were observed. CONCLUSIONS ATTR V122I and AL are equally prevalent as the cause of cardiomyopathy in African Americans referred for a diagnosis of amyloidosis. Available therapy for AL underscores the need for early and accurate determination of amyloid type.

Doxycycline reduces fibril formation in a transgenic mouse model of AL amyloidosis

Systemic AL amyloidosis results from the aggregation of an amyloidogenic immunoglobulin (Ig) light chain (LC) usually produced by a plasma cell clone in the bone marrow. AL is the most rapidly fatal of the systemic amyloidoses, as amyloid fibrils can rapidly accumulate in tissues including the heart, kidneys, autonomic or peripheral nervous systems, gastrointestinal tract, and liver. Chemotherapy is used to eradicate the cellular source of the amyloidogenic precursor. Currently, there are no therapies that target the process of LC aggregation, fibril formation, or organ damage. We developed transgenic mice expressing an amyloidogenic λ6 LC using the cytomegalovirus (CMV) promoter to circumvent the disruption of B cell development by premature expression of recombined LC. The CMV-λ6 transgenic mice develop neurologic dysfunction and Congophilic amyloid deposits in the stomach. Amyloid deposition was inhibited in vivo by the antibiotic doxycycline. In vitro studies demonstrated that doxycycline directly disrupted the formation of recombinant LC fibrils. Furthermore, treatment of ex vivo LC amyloid fibrils with doxycycline reduced the number of intact fibrils and led to the formation of large disordered aggregates. The CMV-λ6 transgenic model replicates the process of AL amyloidosis and is useful for testing the antifibril potential of orally available agents.

Bone Marrow Core Biopsy Specimens in AL (Primary) Amyloidosis A Morphologic and Immunohistochemical Study of 100 Cases

We retrospectively reviewed 100 bone marrow core biopsy specimens from patients with AL (primary) amyloidosis. The morphologic and immunohistochemical features were assessed by standard histochemical stains (H&E, periodic acid-Schiff, Congo red) and immunohistochemical stains for light chain immunoglobulins. Bone marrow core biopsy revealed a plasma cell dyscrasia in 83% (lambda, 65; kappa, 18) of cases. Amyloid deposits were observed in 60% of the bone marrow core biopsy specimens and, when present, were detected most often in blood vessel walls only (39/60). However, if present, interstitial amyloid deposition was significantly more associated with patients with a monoclonal kappa light chain gammopathy (P = .04). Through the careful analysis of standard histochemical and immunohistochemical stains, bone marrow core biopsy provides essential diagnostic information in cases of AL amyloidosis.

Amyloidosis of the gastrointestinal tract: a 13-year, single-center, referral experience

Amyloidosis of the gastrointestinal tract, with biopsy-proven disease, is rare. We reviewed a series of patients who presented with biopsy-proven gastrointestinal amyloidosis and report their clinical characteristics, treatments, and survival. This is a retrospective review of data prospectively collected from January 1998 to December 2011 in a tertiary referral center; 2,334 patients with all types of amyloidosis were evaluated during this period. Seventy-six patients (3.2%) had biopsy-proven amyloid involvement of the gastrointestinal tract. Their median age was 61 years (range, 34-79). Systemic amyloidosis with dominant gastrointestinal involvement was present in 60 (79%) patients, whereas the other 16 (21%) patients had amyloidosis localized to the gastrointestinal tract without evidence of an associated plasma cell dyscrasia or other organ involvement. Of the 60 systemic cases, 50 (83%) had immunoglobulin light-chain, five (8%) had familial lysozyme, three (5%) had wild-type transthyretin, and two (3%) had mutant transthyretin amyloidosis. The most frequent symptoms for all patients were weight loss in 33 (45%) and gastrointestinal bleeding in 27 (36%). Incidental identification of amyloidosis on routine endoscopic surveillance played a role in the diagnosis of seven patients with systemic immunoglobulin light-chain, and four patients with immunoglobulin light-chain localized to the gastrointestinal tract. Amyloid protein subtyping was performed in 12 of the cases of localized disease, and all had lambda light chain disease. Of the 50 patients with systemic immunoglobulin light-chain amyloidosis, 45 were treated with anti-plasma cell therapy. The median survival has not been reached for this group. For the 16 patients with localized gastrointestinal amyloidosis, supportive care was the mainstay of treatment; none received anti-plasma cell therapy. All 16 are alive at a median follow-up of 36 months (range, 1-143). Patients with biopsy-proven gastrointestinal amyloidosis often present with weight loss and bleeding. In localized cases, all that underwent typing were due to lambda light chain amyloidosis and none progressed to systemic disease during the period of follow-up. Most patients with systemic disease had immunoglobulin light-chain, and their tolerance of therapy and median survival were excellent. Although a rare manifestation of amyloidosis, staining for amyloid should be considered in patients undergoing gastrointestinal biopsy who have unexplained chronic gastrointestinal symptoms.

Response of Lymphoepithelial Parotid Cysts to Antiretroviral Treatment in HIV-Infected Adults

Salivary gland enlargement in adults with HIV infection may be caused by viruses, opportunistic pathogens, neoplasms, the Sjogren syndrome, the diffuse infiltrative CD8 lymphocytosis syndrome, or lymphoepithelial parotid cysts [1-14]. The incidence of lymphoepithelial parotid cysts increases in HIV-infected patients, and the cysts are more likely to be chronic, large, bilateral, and multiloculated in these patients than in adults who are not infected with HIV [1, 4-14]. Because most reports of lymphoepithelial cysts in HIV-infected adults have been from retrospective studies of patients who had surgical resection before effective antiretroviral therapy or proper documentation of HIV infection, specific data on the efficacy of current combination antiretroviral therapy are lacking [4, 6-16]. We present data on the antiretroviral treatment of nine HIV-infected patients with chronic lymphoepithelial parotid cysts. Methods Clinical Data Nine HIV-infected patients with chronic lymphoepithelial cysts were followed prospectively between January 1993 to October 1997, with the approval of the human studies committee. All patients had evidence of HIV-1 antibody by enzyme immunoassay and Western blot. CD4 and CD8 lymphocyte counts were measured by flow cytometry, and plasma viral loads were measured by branched-chain DNA assay (Chiron, Emeryville, California). Computed tomography or magnetic resonance imaging was done in eight patients who also had fluid aspirated from at least one parotid cyst. Antiretroviral therapy for HIV infection was administered to all nine patients; five patients also received one or two courses of rapidly tapered prednisone therapy (60 mg daily for 2 days, 40 mg daily for 2 days, 20 mg daily for 2 days, and 10 mg daily for 1 day). Before and after treatment, we clinically estimated cyst size by measuring maximum length and width; the estimated depth of the lesions varied between 5 and 10 cm. Patients with a reduction in gland size of more than 95% and no visible parotid enlargement were considered to have completely responded to medical therapy. Pathologic Evaluation and Immunohistochemical Studies Surgical sections from patient 9 were exposed to lymphocyte cell antigens (CD45), B-cell antigens (CD20, CD79a), T-cell antigens (CD3, CD45Ro), CD68, p24 antigen of HIV (Dako Corp., Carpenteria, California), cytokeratins (AE1/3) and S-100 protein, and cytokeratins (Cam 5.2) and CD1. These antigens were detected by using the strepavidin-horseradish peroxidase detection kit. Results Clinical Data Demographic data, risk behaviors for HIV infection, and CD4 lymphocyte counts are summarized in the Table 1 and Table 2. The patients presented with parotid enlargement that had been present for a mean of 5.4 years (Figure 1, left); four patients also had mild symptoms of dry mouth or dry eyes. In eight patients, computed tomography or magnetic resonance imaging showed bilateral, giant, multiloculated cysts in the intraparotid and periparotid area, with evidence of cervical adenopathy and dense nodular infiltration in and around the parotid gland. Table 1. Characteristics and Management of Nine Patients with Lymphoepithelial Cysts and HIV Infection* Table 2. Table 1. Continued Figure 1. Lymphoepithelial parotid cyst disease in an HIV-infected patient (patient 2) before (left) and after (right) combination antiretroviral therapy. Parotid enlargement was the initial sign of HIV infection in at least seven patients, and all patients reported several previous examinations by health care providers who did not provide a diagnosis, suggest HIV testing, or initiate specific treatment. Related psychosocial reactions, such as anxiety, depression, and reclusiveness, were reported by all patients; six patients who completely responded to medical treatment reported improvement in these problems after therapy. Medical Treatment Although response to medical treatment often varied by the affected side and by patient, all nine patients responded (Table 1 and Table 2). The cysts completely resolved with combination antiretroviral therapy in six patients (Figure 1, right). Of these patients, five received an HIV protease inhibitor and had sustained responses that lasted for a mean of at least 15 months and were associated with increases in CD4 counts and undetectable plasma HIV branched-chain DNA levels. The sixth patient was lost to follow-up 5 months later. Four of the six patients also received one or two short courses of prednisone therapy that produced a rapid and dramatic reduction in parotid gland size without side effects. Eight patients also had cyst aspiration, which was well tolerated but usually provided only temporary and limited improvement. Partial responses followed by relapses were noted in the three patients who were poorly compliant with antiretroviral therapy. Pathologic Evaluation and Immunohistochemical Studies Six months after antiretroviral therapy was stopped, a cystic parotid mass that measured 7 4 3 cm and contained three separate cysts (which ranged from 1.8 to 2.5 cm in diameter) was removed from patient 9. The cysts were distinct from the normal parotid tissue and were lined by both keratinizing and nonkeratinizing squamous epithelium, which was variably infiltrated by small lymphocytes. Adjacent dense lymphoid tissue showed various degrees of follicular hyperplasia, occasional secondary follicle lysis, and multinucleated giant cells. The interfollicular areas were notable for marked proliferation of plasma cells and prominent postcapillary venules. Immunoperoxidase studies of the cyst lining yielded positive results for cytokeratin, which confirmed the cysts epithelial origin. The lymphoid component was positive for CD45, and B cells (CD20, CD79a) were localized in secondary follicles and T cells (CD3, CD45Ro) that stained the paracortical and intrafollicular areas. Lymphocytes that infiltrated the cyst epithelial lining stained predominantly as B cells. Antisera to the HIV p24 antigen localized the virus to germinal centers of secondary follicles and had an interstitial or dendritic pattern. Large multinucleated cells that were present in germinal centers and paracortical areas also stained strongly positive for HIV p24 antigen and expressed CD68 antigen (Kp1); this is consistent with a cell of histiocytic or macrophage lineage. Langerhans cells were occasionally present in paracortical areas. Discussion Our data suggest that lymphoepithelial cysts are not widely recognized as a manifestation of HIV disease. This lack of recognition results in delayed diagnosis and treatment [4, 12]. Patients with benign lymphoepithelial cysts are usually asymptomatic and have CD4 lymphocyte counts that range from 300 to 600 cells/mm3 [4, 9, 11-13]. The cysts are usually chronic, large, multiloculated, and bilateral [4, 12, 13]. Parotid cysts may be diagnosed clinically and confirmed by computed tomography or magnetic resonance imaging [1, 13]. Although lymphoepithelial cysts are highly suggestive of HIV infection, they may be seen with the Sjogren syndrome, the diffuse infiltrative CD8 lymphocytosis syndrome, or tumors or opportunistic infections of the parotid gland [1-3]. In the absence of HIV infection or for patients who have unusual clinical findings or inadequate response to medical therapy, fine-needle aspiration biopsy and surgical resection should be considered. Previous studies of lymphoepithelial cysts often lacked proper documentation of HIV infection, focused on surgical treatment, and provided limited data on the efficacy of antiretroviral therapy [4-14]. Shaha and coworkers [11] reported a complete response in one of two patients treated with zidovudine, and Schiodt and coworkers [13] reported regression of parotid cysts in four patients treated with zidovudine and steroids. Terry and coworkers [4] reported a complete response in one of six patients treated with zidovudine and a partial response in two patients treated with zidovudine and radiation. Low-dose radiation therapy may provide temporary cosmetic palliation of benign lymphoepithelial cysts [17]. Treatment of HIV disease has improved dramatically with the recent increase in our understanding of HIV dynamics and pathogenesis and progress in the use of viral load assays and combination antiretroviral therapy [15, 16, 18]. In our series, complete, sustained resolution of parotid cyst disease was associated with continued compliance with combination antiretroviral therapy, undetectable HIV branched-chain DNA levels, and increases in CD4 lymphocyte counts. Therefore, for HIV-infected patients with lymphoepithelial cysts, we advocate the currently recommended treatment of combination antiretroviral therapy that includes a protease inhibitor [15, 16]. Although corticosteroid therapy (probably because of its anti-inflammatory and lympholytic effects) resulted in rapid, dramatic decreases in the size of parotid cysts, it may be unnecessary for patients receiving highly active antiretroviral combination therapy [13]. Cyst aspiration was helpful in patients with large cysts that did not regress with antiretroviral therapy but, when used alone, it yielded only temporary improvement. Bernier and Bhaskar [5] hypothesized that lymphoepithelial cysts in patients without HIV infection arise from epithelial ductular inclusions in lymph nodes. The increased incidence of lymphoepithelial cysts and the different clinical presentation associated with HIV infection probably reflect the high concentrations and rapid turnover of HIV in hyperplastic lymphoid tissue in and adjacent to the parotid parenchyma [1, 5, 18]. Our immunohistologic studies showed high concentrations of HIV p24 antigen in lymphoid tissue; this confirmed the earlier reports of HIV-infected lymph tissues and the immunohistochemical analysis of a patient with benign lymphoepithelial cysts [19, 20]. We hypothesize that HIV-induced cytokines and lymphoid hyperplasia stimulate the adjacent ductal

Matrix Metalloproteinases and Their Tissue Inhibitors in Cardiac Amyloidosis Relationship to Structural, Functional Myocardial Changes and to Light Chain Amyloid Deposition

Background—Cardiac amyloidosis is characterized by amyloid infiltration resulting in extracellular matrix disruption. Amyloid cardiomyopathy due to immunoglobulin light chain protein (AL-CMP) deposition has an accelerated clinical course and a worse prognosis compared with non-light chain cardiac amyloidoses (ie, forms associated with wild-type or mutated transthyretin [TTR]). We therefore tested the hypothesis that determinants of proteolytic activity of the extracellular matrix, the matrix metalloproteinases (MMPs), and their tissue inhibitors (TIMPs) would have distinct patterns and contribute to the pathogenesis of AL-CMP versus TTR-related amyloidosis. Methods and Results—We studied 40 patients with systemic amyloidosis: 10 AL-CMP patients, 20 patients with TTR-associated forms of cardiac amyloidosis, ie, senile systemic amyloidois (involving wild-type TTR) or mutant TTR, and 10 patients with AL amyloidosis without cardiac involvement. Serum MMP-2 and -9, TIMP-1, -2, and -4, brain natriuretic peptide values, and echocardiography were determined. AL-CMP and TTR-related amyloidosis groups had similar degrees of increased left ventricular wall thickness. However, brain natriuretic peptide, MMP-9, and TIMP-1 levels were distinctly elevated accompanied by marked diastolic dysfunction in the AL-CMP group versus no or minimal increases in the TTR-related amyloidosis group. Brain natriuretic peptide, MMPs, and TIMPs were not correlated with the degree of left ventricular wall thickness but were correlated to each other and to measures of diastolic dysfunction. Immunostaining of human endomyocardial biopsies showed diffuse expression of MMP-9 and TIMP-1 in AL-CMP and limited expression in TTR-related amyloidosis hearts. Conclusions—Despite comparable left ventricular wall thickness with TTR-related cardiac amyloidosis, AL-CMP patients have higher brain natriuretic peptide, MMPs, and TIMPs, which correlated with diastolic dysfunction. These findings suggest a relationship between light chains and extracellular matrix proteolytic activation that may play an important role in the functional and clinical manifestations of AL-CMP, distinct from the other non-light chain cardiac amyloidoses.Background— Cardiac amyloidosis is characterized by amyloid infiltration resulting in extracellular matrix disruption. Amyloid cardiomyopathy due to immunoglobulin light chain protein (AL-CMP) deposition has an accelerated clinical course and a worse prognosis compared with non–light chain cardiac amyloidoses (ie, forms associated with wild-type or mutated transthyretin [TTR]). We therefore tested the hypothesis that determinants of proteolytic activity of the extracellular matrix, the matrix metalloproteinases (MMPs), and their tissue inhibitors (TIMPs) would have distinct patterns and contribute to the pathogenesis of AL-CMP versus TTR-related amyloidosis. Methods and Results— We studied 40 patients with systemic amyloidosis: 10 AL-CMP patients, 20 patients with TTR-associated forms of cardiac amyloidosis, ie, senile systemic amyloidois (involving wild-type TTR) or mutant TTR, and 10 patients with AL amyloidosis without cardiac involvement. Serum MMP-2 and -9, TIMP-1, -2, and -4, brain natriuretic peptide values, and echocardiography were determined. AL-CMP and TTR-related amyloidosis groups had similar degrees of increased left ventricular wall thickness. However, brain natriuretic peptide, MMP-9, and TIMP-1 levels were distinctly elevated accompanied by marked diastolic dysfunction in the AL-CMP group versus no or minimal increases in the TTR-related amyloidosis group. Brain natriuretic peptide, MMPs, and TIMPs were not correlated with the degree of left ventricular wall thickness but were correlated to each other and to measures of diastolic dysfunction. Immunostaining of human endomyocardial biopsies showed diffuse expression of MMP-9 and TIMP-1 in AL-CMP and limited expression in TTR-related amyloidosis hearts. Conclusions— Despite comparable left ventricular wall thickness with TTR-related cardiac amyloidosis, AL-CMP patients have higher brain natriuretic peptide, MMPs, and TIMPs, which correlated with diastolic dysfunction. These findings suggest a relationship between light chains and extracellular matrix proteolytic activation that may play an important role in the functional and clinical manifestations of AL-CMP, distinct from the other non–light chain cardiac amyloidoses. Received April 25, 2008; accepted September 23, 2008.

Detection of Impaired Homologous Recombination Repair in NSCLC Cells and Tissues

Introduction: Homologous recombination repair (HRR) is a critical pathway for the repair of DNA damage caused by cisplatin or poly-ADP ribose polymerase (PARP) inhibitors. HRR may be impaired by multiple mechanisms in cancer, which complicates assessing the functional HRR status in cells. Here, we monitored the ability of non–small-cell lung cancer (NSCLC) cells to form subnuclear foci of DNA repair proteins as a surrogate of HRR proficiency. Methods: We assessed clonogenic survival of 16 NSCLC cell lines in response to cisplatin, mitomycin C (MMC), and the PARP inhibitor olaparib. Thirteen tumor explants from patients with NSCLC were subjected to cisplatin ex vivo. Cells were assayed for foci of repair-associated proteins such as BRCA1, FANCD2, RAD51, and &ggr;-H2AX. Results: Four cell lines (25%) showed an impaired RAD51 foci-forming ability in response to cisplatin. Impaired foci formation correlated with cellular sensitivity to cisplatin, MMC and olaparib. Foci responses complemented or superseded genomic information suggesting alterations in the ATM/ATR and FA/BRCA pathways. Because baseline foci in untreated cells did not predict drug sensitivity, we adapted an ex vivo biomarker assay to monitor damage-induced RAD51 foci in NSCLC explants from patients. Ex vivo cisplatin treatment of explants identified two tumors (15%) exhibiting compromised RAD51 foci induction. Conclusions: A fraction of NSCLC harbors HRR defects that may sensitize the affected tumors to DNA-damaging agents including PARP inhibitors. We propose that foci-based functional biomarker assays represent a powerful tool for prospective determination of treatment sensitivity, but will require ex vivo techniques for induction of DNA damage to unmask the underlying HRR defect.

Preclinical development of siRNA therapeutics for AL amyloidosis

Amyloid light chain (AL) amyloidosis is a rare hematologic disorder characterized by the accumulation of a misfolded monoclonal immunoglobulin (Ig) light chain (LC) as fibrillar protein deposits. Current treatments, including cytotoxic chemotherapy and immunomodulatory therapy, are directed at killing the plasma cells that produce the LCs, but have significant toxicity for other cell types. We have designed small interfering RNAs (siRNAs) targeting the amyloidogenic LC messenger RNA (mRNA) in order to reduce expression of the amyloid precursor protein. Using nanomolar concentrations of siRNAs, we have inhibited synthesis of LC in transfected cells in vitro in a dose-dependent fashion. Furthermore, in an in vivo plasmacytoma mouse model of AL amyloidosis, we have demonstrated that these siRNAs can significantly reduce local production and circulating levels of LC. This model system highlights the therapeutic potential of siRNA for AL amyloidosis.

Cardiac nonamyloidotic immunoglobulin deposition disease

Cardiac nonamyloidotic immunoglobulin (Ig) deposition disease (CIDD) is a rare disorder characterized by Ig deposition in the myocardium associated with plasma cell dyscrasias. A retrospective review of cardiac biopsies performed at two different institutions identified eight patients with CIDD. All patients had plasma cell dyscrasias with monoclonal gammopathy. Three had IgG λ, two had IgG κ, one had IgD κ and one each had free κ and free λ light chain. Four patients had concurrent amyloidosis involving other organs. One had amyloidosis of kidney alone, one had amyloidosis of kidney and abdominal fat pad and two others had amyloidosis of bone marrow vasculature. Three patients had dialysis-dependent renal insufficiency. None of the patients had symptoms of heart failure. Six patients had echocardiographically demonstrable concentric left ventricular hypertrophy with diastolic dysfunction. Two patients had significant cardiac arrhythmias requiring medical intervention. On endomyocardial biopsy, all eight had normal appearing myocardium on light microscopy with negative Congo Red and Thioflavin T stains. On immunofluorescent staining of the cardiac biopsies, all eight stained positive for interstitial Ig deposition. Electron microscopy (EM) confirmed the presence of granular deposits of Igs in the myocardium in five of the eight patients. EM studies were not available in one patient and two others had normal EM studies. In conclusion, CIDD should be considered in the spectrum of cardiovascular pathology in patients with plasma cell dyscrasias. They often, but not always, have left ventricular hypertrophy. These patients may be at risk for developing arrhythmias as well as diastolic dysfunction. Unless immunofluorescent and EM studies are performed routinely in biopsy material, this entity may be missed in the absence of amyloidosis. Concurrent amyloidosis in other organs sheds a unique perspective into the role of local microenvironment in the pathogenesis of systemic Ig deposition disease and amyloidosis.